Anticoccidial fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene recombination and the field of medicaments, and provides an anticoccidial fusion protein, the amino acid sequence of which is formed by sequentially connecting a broad-spectrum antiprotozoon polypeptide APP amino acid sequence, an oxidative pressure sensitive Linker amino acid sequence, a flexible Linker amino acid sequence and a lignin peroxidase mutant LiPM amino acid sequence; the amino acid sequences of the broad-spectrum antiprotozoon polypeptide APP, the oxidative pressure sensitive Linker, the flexible Linker and the lignin peroxidase mutant LiPM are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 4. The invention also provides a preparation method and application of the anticoccidial fusion protein. The method is designed based on APP and LiPM, when the method is used as an insecticide, the outer layer of the oocyst wall can be subjected to enzymolysis through LiPM, meanwhile, the APP enters the inner layer of the oocyst to effectively kill the oocyst, and a better anti-coccidiosis effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical fields of gene recombination and pharmaceuticals, and particularly to an anti-coccidia fusion protein, a preparation method thereof, and an application thereof. Background Art

[0002] Our company's prior patent CN 116903753B discloses a broad-spectrum anti-protozoal polypeptide, the amino acid sequence of which is sequentially connected by an HIV-TAT transduction protein, a flexible Linker protein, and a mutant APLV4-VP protein. This polypeptide shows an effect equivalent to that of antibiotic drugs in the application of broad-spectrum anti-protozoa, and does not cause drug resistance, providing a safe and effective non-antibiotic anti-protozoal drug for the aquaculture industry. However, farmers have reported that this drug is only significantly effective against protozoa other than coccidia, and the condition is likely to relapse after treating coccidia infection.

[0003] The HIV-TAT transduction protein is a small molecule protein that can cross the host cell membrane through its special cell-penetrating peptide (CPP) structure, usually interacting with lipids on the cell membrane, or guiding the protein it carries into the cell through the endocytic pathway and playing an important role. The coccidian oocyst is an important transmission structure in the coccidia life cycle. The coccidian oocyst consists of an inner and an outer wall structure, and the inner and outer layers have different functions and components. The inner layer is similar to an ordinary cell membrane and is mainly composed of protein, lipid, and polysaccharide complexes. The outer layer is composed of collagen-like proteins and keratin cross-linked by tyrosine, forming a strong three-dimensional network structure, which causes the HIV-TAT transduction protein to be unable to transport the mutant APLV4-VP protein through the outer oocyst membrane and reach the inside of the egg to play a killing role. In the middle and late stages of coccidia infection, the oocysts have already formed. Therefore, applying the solution described in patent CN 116903753B still cannot effectively kill these oocysts in vivo. After the oocysts are discharged from the body, if the drug is stopped, the ingestion of the oocysts will cause re-infection within the population.

[0004] A fusion protein is a novel protein with multiple biological activities that is precisely linked at the amino acid level by gene recombination technology, integrating specific functional regions or complete proteins derived from different proteins. This fusion strategy realizes the synergistic integration of different protein functional domains, enabling the recombinant fusion protein to exhibit more superior biological characteristics compared to single proteins. In scientific fields such as medicine, biotechnology, and vaccine research and development, recombinant fusion proteins exhibit great application potential and value, such as being used to develop new anti-tumor, anti-rheumatic, and autoimmune disease treatment drugs.

[0005] Accordingly, the present invention attempts to combine fusion protein technology to develop a medicament with better anti-coccidia effects, especially with a better killing effect on coccidian oocysts. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an anti-coccidia fusion protein, its preparation method and application. It is designed based on a broad-spectrum anti-protozoon polypeptide (APP) and a lignin peroxidase mutant (LiPM). When used as an insecticide, LiPM can enzymatically hydrolyze the outer layer of the oocyst wall, and at the same time, APP can enter the inner layer of the egg to effectively kill the eggs, achieving a better anti-coccidia effect.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] An anti-coccidia fusion protein, the amino acid sequence of the anti-coccidia fusion protein is sequentially connected by the amino acid sequence of a broad-spectrum anti-protozoon polypeptide (APP), an oxidative stress-sensitive Linker amino acid sequence, a flexible Linker amino acid sequence, and a lignin peroxidase mutant (LiPM) amino acid sequence; the amino acid sequences of the broad-spectrum anti-protozoon polypeptide (APP), the oxidative stress-sensitive Linker, the flexible Linker, and the lignin peroxidase mutant (LiPM) are shown in SEQ ID NO.1-4 respectively.

[0009] As one of the preferred embodiments of the present invention, the lignin peroxidase mutant LiPM is specifically mutated from lignin peroxidase LiP shown by the sequence of SEQ ID NO.5, and the mutation sites are H at position 179 → A at position 179 and T at position 180 → F at position 180 of SEQ ID NO.5.

[0010] A preparation method of the above anti-coccidia fusion protein, comprising the following steps:

[0011] (1) Obtain the amino acid sequences of APP protein, oxidative stress-sensitive Linker, flexible Linker, and LiPM protein respectively;

[0012] (2) Connect the APP protein and the LiPM protein using the oxidative stress-sensitive Linker and the flexible Linker to obtain the amino acid sequence of the anti-coccidia fusion protein, as shown in SEQ ID NO.6;

[0013] (3) Use DNAMAN software, combined with the codon preference of Saccharomyces cerevisiae for optimization, reverse translate the amino acid sequence of the anti-coccidia fusion protein, and add the restriction enzyme site EcoRⅠ and the start codon upstream, and add the stop codon and the restriction enzyme site NotⅠ downstream to obtain the nucleic acid sequence of the anti-coccidia fusion protein, as shown in SEQ ID NO.7;

[0014] (4) Use restriction enzymes to perform double digestion on the fusion protein gene sequence and the expression plasmid respectively. After ligating the digested products, transform them into cloning bacteria; screen positive clones, extract plasmids and then perform sequencing verification; linearize the verified plasmids and then introduce them into expression bacteria; pick high-copy transformants for culture to obtain the target yeast engineering bacteria;

[0015] (5) Ferment and purify the anti-coccidia fusion protein.

[0016] As one of the preferred embodiments of the present invention, in the step (5), the specific steps of fermentation and purification are as follows:

[0017] Pick monoclonal colonies growing on the bleomycin plate and inoculate them into a conical flask containing BMGY medium, culture overnight as the seed solution; inoculate the seed solution into a conical flask containing BMGY medium, culture until the logarithmic growth phase, and add methanol for induction; thereafter, regularly add methanol to the medium to maintain continuous expression of induction; after the induction expression is completed, centrifuge to retain the supernatant, and perform protein precipitation, centrifugation, suspension, and dialysis on the supernatant to obtain the target protein.

[0018] An application of the above anti-coccidia fusion protein in the preparation of anti-coccidia insecticides.

[0019] As one of the preferred embodiments of the present invention, the anti-coccidia fusion protein acts by enzymatically degrading the outer layer of the oocyst wall of coccidia through LiPM, and APP enters the inner layer of the oocysts to eliminate the oocysts.

[0020] Design principle of the fusion protein of the present invention:

[0021] Lignin peroxidase (LiP) can enzymatically degrade lignin, mainly recognize the benzene ring structure and break the C-C bond. The oocyst wall of coccidia oocysts is mainly formed by tyrosine cross-linking to form di-tyrosine bonds that are difficult to be enzymatically degraded by digestive enzymes such as trypsin. Its structure has a benzene ring structure that can be recognized by LiP, and the di-tyrosine bond is C-C. Therefore, lignin peroxidase has the possibility of enzymatically degrading di-tyrosine bonds. However, in order to make lignin peroxidase better enzymatically degrade di-tyrosine bonds, the binding center of LiP - the heme-binding residue site can be mutated. Specifically, the present invention selects the mature peptide sequence of LiP from Coriolus versicolor (NCBI accession number AAA34049, amino acid sequence as shown in SEQ ID NO.5), and mutates its 179th H to A and 180th T to F. The amino acid sequence after mutation is as shown in SEQ ID NO.3, denoted as lignin peroxidase mutant LiPM.

[0022] Therefore, applying LiPM to the breaking of the oocyst wall of coccidia can effectively solve the problem that coccidia eggs cannot be broken by enzymatic hydrolysis under conventional conditions. Further combining with the broad-spectrum anti-protozoal polypeptide (denoted as APP) disclosed in our company's prior patent CN 116903753B can enable APP to enter the inner layer of the eggs and effectively kill the eggs.

[0023] The advantages of the present invention compared with the prior art are as follows:

[0024] (1) The present invention is designed and prepared into a fusion protein based on the broad-spectrum anti-protozoal polypeptide APP and the lignin peroxidase mutant LiPM; when used as an insecticide, LiPM of the fusion protein can effectively enzymatically hydrolyze the outer layer of the oocyst wall. Subsequently, APP can enter the inner layer of the eggs to effectively kill the eggs, achieving a better anti-coccidia effect;

[0025] (2) The fusion protein of the present invention uses an oxidative stress-sensitive Linker and a flexible Linker as the connecting peptides. Among them, the flexible Linker can space APP and LiPM spatially and does not affect their respective biological functions; while the oxidative stress-sensitive Linker breaks when LiPM enzymatically hydrolyzes the di-tyrosine bond, separating APP, so that it can enter the inner layer of the oocyst more effectively and quickly;

[0026] (3) The present invention uses Pichia pastoris for fermentation and expression. Compared with prokaryotic expression, its structure is more advanced and has better activity, and the post-treatment process of the product is simple, with low cost and can be widely used. Description of the Drawings

[0027] Figure 1 is the Page electrophoresis diagram of the FACP and APP-LiP proteins of the present invention (in the figure, M: Protein Marker 26610; Lane 1: empty bacteria control; Lane 2: FACP protein, Lane 3: APP-LiP protein);

[0028] Figure 2 is the microscopic result diagram of the FACP and APP-LiP proteins of the present invention enzymatically hydrolyzing coccidia eggs (in the figure, Figure A is the microscopic examination diagram after APP-LiP enzymatic hydrolysis; Figure B is the microscopic examination diagram after FACP enzymatic hydrolysis; 400×). Detailed Embodiments

[0029] The embodiments of the present invention will be described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and the detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, the experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are conventional materials directly purchased from existing biochemical reagent stores unless otherwise specified.

[0030] Example 1. Design of the anti-coccidia fusion protein:

[0031] The amino acid sequence of the anti-coccidia fusion protein is composed of the amino acid sequence of a broad-spectrum anti-protozoal polypeptide (APP) (SEQ ID NO.1), the amino acid sequence of an oxidative stress-sensitive Linker (SEQ ID NO.2), the amino acid sequence of a flexible Linker (SEQ ID NO.3), and the amino acid sequence of a lignin peroxidase mutant (LiPM) (SEQ ID NO.4) connected in sequence.

[0032] Among them, the lignin peroxidase mutant (LiPM) is mutated from the lignin peroxidase (LiP) shown by the SEQ ID NO.5 sequence, and the mutation sites are H at position 179 → A at position 179 and T at position 180 → F at position 180 of SEQ ID NO.5.

[0033] The finally formed anti-coccidia fusion protein in this example, denoted as FACP (Fusion Anticoccidial Peptide with High Efficacy Against Coccidia), has the complete amino acid sequence as shown in SEQ ID NO.6.

[0034] Example 2. Obtaining the nucleic acid sequence of the anti-coccidia fusion protein:

[0035] Using the DNAMAN software and optimizing in combination with the codon preference of Saccharomyces cerevisiae, the amino acid sequence of the anti-coccidia fusion protein was reverse-translated, and the restriction enzyme site EcoRⅠ and the start codon were added upstream, and the stop codon and the restriction enzyme site NotⅠ were added downstream to obtain the nucleic acid sequence of the anti-coccidia fusion protein, as shown in SEQ ID NO.7.

[0036] Example 3. Preparation of the engineering bacteria of the anti-coccidia fusion protein:

[0037] The nucleic acid sequence shown in SEQ ID NO.7 was sent to BGI for synthesis into the pT19 cloning plasmid.

[0038] Using the chemical transformation method, the FACP-pT19 plasmid was transferred into DH5α cloning bacteria, coated on an ampicillin-resistant LB plate, and cultured overnight. Positive clones were picked and cultured in an ampicillin-resistant LB liquid medium at 37°C and 220 rpm for 8 h, and the strains were preserved, and the remaining bacterial liquid was cultured overnight.

[0039] The plasmid was extracted using the AbLab plasmid extraction kit. To avoid mutations caused by PCR, the extracted plasmid and the purchased pPiczαA plasmid were directly double-digested with the restriction enzymes EcoRⅠ and NotⅠ.

[0040] Take the target gene segment after digestion with pT19 and the pPiczαA backbone segment, perform gel extraction and recovery on both, ligate the two recovered products using Takara's T4 ligase, and then transfer them into DH5α cloning bacteria by chemical transformation method.

[0041] Screen positive clones using an LB plate containing 25 μg / mL bleomycin. Pick positive clones into an LB liquid medium containing 25 μg / mL bleomycin, culture at 37 °C and 220 rpm for 8 h, preserve the seeds, and continue to culture the remaining bacterial liquid overnight. After plasmid extraction, send it to BGI for sequencing verification.

[0042] Linearize the verified plasmid using SacⅠ endonuclease.

[0043] Take the X33 yeast cells with an OD 600 reaching 0.6 - 1.0, centrifuge at 4000 rpm for 10 min, discard the supernatant, wash the cells twice with ice-cold 1 M LiAc solution to remove the salts in the medium. Suspend the washed yeast cells in 1 M LiCl and perform transformation under the following electroporation conditions: voltage: 2.0 kV / cm; pulse width: 5 ms; capacitance: 100 μF; conductivity: 800 Ω.

[0044] After electroporation, transfer the cells to a liquid medium containing 1 M SOS, incubate at 30 °C for 1 h, then centrifuge at 4000 rpm for 10 min to collect the precipitate, add 200 μL of sterile water to resuspend. Spread it on a YPDS plate containing 1 mg / mL bleomycin, invert the plate and place it in a 30 °C incubator for 5 days. Finally, pick positive transformants and culture them under constant temperature conditions to obtain the target yeast engineering bacteria, denoted as FACP-X33.

[0045] Example 4: Fermentation and purification of anti-coccidia fusion protein

[0046] Take FACP-X33 and streak it in four zones on a YPDS plate containing 1 mg / mL bleomycin, invert the plate and place it in a 30 °C incubator for 5 days. Pick positive transformants and inoculate them into a 50 ml conical flask containing 20 ml of BMGY medium, culture at 30 °C and 220 r / min overnight until the OD 600 reaches 4, and use it as the seed solution, inoculate it into a 2 L conical flask containing 1 L of BMGY medium, culture at 30 °C and 250 r / min for about 20 h until the OD 600 reaches 30, then add 0.5% v / v methanol for induction, set the temperature to 28 °C, and set the rotation speed to 220 r / min. Thereafter, add 0.5% methanol to the medium every 24 h to maintain the continuous expression of induction. Stop the induction expression after 48 h.

[0047] At room temperature, centrifuge at 4000 r / min for 10 min and retain the supernatant. Add saturated ammonium sulfate to the supernatant after centrifugation at a ratio of 1:1. Then place the mixture in a refrigerator at 4°C and let it stand for 24 h to precipitate the protein in the fermentation broth by flocculation. Centrifuge at 12000 rpm for 15 min at 4°C, collect the precipitate, and discard the supernatant. Suspend the precipitate with an appropriate amount of PBS buffer and dialyze it with PBS of the same concentration as the suspension to remove salts, thus obtaining the target protein FACP. As Figure 1 shown in Lane 2.

[0048] Comparative Example 1: Preparation of an anti-coccidia fusion protein without mutation of LiP:

[0049] Design an anti-coccidia fusion protein without mutation of LiP, and the amino acid sequence of this protein is arranged as: APP (SEQ ID NO.1) + oxidative stress-sensitive Linker (SEQ ID NO.2) + flexible Linker (SEQ ID NO.3) + LiP (SEQ ID NO.5). The finally obtained anti-coccidia fusion protein without mutation of LiP has an amino acid sequence as shown in SEQ ID NO.8, and the corresponding nucleic acid sequence obtained according to the method of Example 2 is as shown in SEQ ID NO.9.

[0050] Furthermore, referring to the methods of Examples 3 and 4 above, construct an engineering bacterium for the designed "anti-coccidia fusion protein without mutation of LiP" and express and purify the protein to obtain the corresponding target protein, denoted as APP-LiP, as Figure 1 shown in Lane 3.

[0051] Experimental Example 1: Verification of enzymatic hydrolysis of coccidia eggs:

[0052] Imitate the pH of the chicken small intestine cecum segment, and use PBS with pH 6.0 to dissolve the anti-coccidia fusion protein (FACP) of the present invention and the anti-coccidia fusion protein without mutation of lignin peroxidase in Comparative Example 1 (APP-LiP) respectively, so that the concentration of both is 0.1 mg / mL. Take 10 mL of each dissolved protein solution and add 1 mL of coccidia eggs at a concentration of 10,000 eggs / mL, so that the final concentration of the eggs is about 1000 eggs / mL, and make 3 replicates.

[0053] Imitate the chicken body temperature and gastrointestinal transit time, place it in a constant temperature water bath shaker at 41°C and incubate for 4 h, and then observe and count the intact eggs under the microscope.

[0054] The results are shown in Table 1 and Figure 2 as shown. Figure 2 In it, in Figure A, the eggs are still intact and the membrane structure is clearly visible. In Figure B, the egg sac of the eggs ruptures and sporozoites are released.

[0055] Table 1 Effect of FACP and APP-LiP on enzymatic hydrolysis of eggs

[0056] Group FACP APP-LiP Mean number of intact eggs 4526 10000 Percentage of eggs with ruptured membranes 54.7％ 0％

[0057] From Table 1 and Figure 2 the results show that APP-LiP does not have the function of enzymatically degrading eggs or the function of breaking the membrane after enzymatically degrading eggs, while FACP has a certain function of enzymatically degrading eggs.

[0058] Experimental Example 2. Verification of the anti-coccidial effect of the anti-coccidial fusion protein:

[0059] (1) Drug treatment

[0060] After freeze-drying the anti-coccidial fusion protein (FACP), the test dose was 100 grams of effective additive per ton of feed; after freeze-drying the broad-spectrum anti-protozoal polypeptide (APP), the test dose was 100 grams of effective additive per ton of feed.

[0061] For spraying, FACP used PBS as a diluent to prepare a 10% FACP protein solution, added 10% glycerol to maintain humidity, added 2% saponin to improve uniformity, and stored it at 4°C for later use after mixing evenly.

[0062] (2) Coccidia challenge and treatment

[0063] Thirty-day-old white - feather chickens free of Eimeria infection were selected as experimental animals. They were grouped according to Table 2. There were 24 chickens in each group of the FACP continuous treatment for 7 days group, and 36 chickens in each of the other ordinary groups. Each group was raised in a flat - raising manner in an environment free of coccidia contamination. Each chicken was orally administered 10,000 Eimeria oocysts. The drinking water, feed intake, feces, and deaths of chickens in each group were observed and recorded daily. Dead chickens were dissected in a timely manner to analyze the causes of death. Four days after inoculation with the parasite, medicated feed was started. The drinking water, feed intake, feces, and deaths of chickens in each group were observed and recorded daily. Dead chickens were dissected in a timely manner to analyze the causes of death. After 3 days of medication, the medication was stopped. Ten surviving chickens from each group were decapitated and sacrificed. Their weights were measured. The ceca were taken, and the cecal mucosa and contents were scraped to detect and calculate the number of oocysts per chicken. In the original environment, the chickens were continued to be fed with blank feed without added drugs for 5 days. Ten surviving chickens from each group were decapitated and sacrificed. Their weights were measured. The ceca were taken, and the cecal mucosa and contents were scraped to detect and calculate the number of oocysts per chicken. In the original environment, the chickens were continued to be fed with feed added with drugs for 10 days. Ten surviving chickens from each group were decapitated and sacrificed. Their weights were measured. The ceca were taken, and the cecal mucosa and contents were scraped to detect and calculate the number of oocysts per chicken. In the FACP continuous treatment group (including the group of FACP continuous treatment for 7 days during inoculation with the parasite + the group of spraying FACP in the farm), 4 days after inoculation with the parasite, FACP was continuously used for treatment for 7 days, and then the drug was stopped and the chickens were switched to blank feed for continued feeding. Ten surviving chickens were decapitated and sacrificed 7 days after treatment. Their weights were measured. The ceca were taken, and the cecal mucosa and contents were scraped to detect and calculate the number of oocysts per chicken. And 8 days later, ten surviving chickens were decapitated and sacrificed together with other groups. Their weights were measured. The ceca were taken, and the cecal mucosa and contents were scraped to detect and calculate the number of oocysts per chicken. The environment was disinfected by spraying FACP 2 times a day until the end of the experiment for the corresponding group.

[0064] Table 2 Grouping of coccidia inoculation and treatment

[0065]

[0066]

[0067] (3) Evaluation of anticoccidial effect

[0068] The anticoccidial index (ACI) was set. ACI=(survival rate + relative weight gain rate)-(lesion value + oocyst value); survival rate=(number of surviving chickens at the end of the experiment / number of experimental chickens)×100%; relative weight gain rate=(weight gain of the experimental group / weight gain of the blank control group)×100%; If ACI≥160, it was judged as excellent; if ACI = 130 - 159, it was judged as good; if ACI = 100 - 129, it was judged as low - sensitivity; if ACI<100, it was judged as ineffective. The results of the anticoccidial index (ACI) of each experimental group are shown in Table 3.

[0069] Table 3 Anticoccidial index (ACI) of each experimental group

[0070]

[0071]

[0072] In this experiment, the dose of infecting chickens with parasites per chicken was a relatively low dose. However, the use of the drug in this experiment started from the 4th day after infecting with parasites. At this time, the first-generation eggs had begun to be produced in the chickens of each group infected with parasites. From the perspective of the anti-protozoan index (ACI), the ACI values of groups 6-9 after continuously using FACP for 7 days were: 147.1, 170.4, 171.7, 187.4, all showing effects above good. In particular, after discontinuing the drug for 8 days after continuously using it for 7 days in groups 8 and 9, no reinfection occurred, and the ACI values were both above 200, with outstanding effects. From the comparison between the groups sprayed with FACP and those not sprayed with FACP, spraying with FACP may have a certain effect of killing the eggs in the venue, thus helping each drug to achieve better effects. Accordingly, the present invention provides a fusion protein for anti-coccidiosis that has a better effect than the "broad-spectrum anti-protozoan polypeptide".

[0073] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anticoccidial fusion protein, characterized in that The amino acid sequence of the anticoccidial fusion protein is composed of a broad-spectrum antiprotozoan polypeptide APP amino acid sequence, an oxidative stress-sensitive Linker amino acid sequence, a flexible Linker amino acid sequence and a lignin peroxidase mutant LiPM amino acid sequence connected in sequence; the amino acid sequences of the broad-spectrum antiprotozoan polypeptide APP, the oxidative stress-sensitive Linker, the flexible Linker and the lignin peroxidase mutant LiPM are shown in SEQ ID NOs. 1 to 4, respectively.

2. The anticoccidial fusion protein according to claim 1, characterized in that The lignin peroxidase mutant LiPM is specifically derived from the mutation of the lignin peroxidase LiP shown in the sequence of SEQ ID NO.5, and the mutation sites are 179th H→179th A and 180th T→180th F of SEQ ID NO.

5.

3. A method for preparing anticoccidial fusion protein according to claim 1 or 2, characterized in that: The steps include: (1) Obtain the amino acid sequences of the broad-spectrum antiprotozoal peptide APP, oxidative stress-sensitive linker, flexible linker, and lignin peroxidase mutant LiPM; (2) Using an oxidative stress-sensitive linker and a flexible linker to connect the APP protein and the LiPM protein, the amino acid sequence of the anti-coccidian fusion protein is obtained, as shown in SEQ ID NO.6; (3) Using DNAMAN software, combined with Saccharomyces cerevisiae codon preference optimization, the amino acid sequence of the anti-coccidial fusion protein was reverse translated, and the restriction site EcoRⅠ and the start codon were added upstream, and the stop codon and the restriction site NotⅠ were added downstream to obtain the nucleic acid sequence of the anti-coccidial fusion protein, as shown in SEQ ID NO.7; (4) Double-digest the fusion protein gene sequence and the expression plasmid using endonucleases, connect the digestion products and transform them into cloning bacteria; screen positive clones, extract plasmids and perform sequencing verification; linearize the verified plasmids and then introduce them into the expression bacteria; select high-copy transformants for culture to obtain the target yeast engineering bacteria; (5) Fermentation and purification of anticoccidian fusion protein.

4. The method for preparing anticoccidial fusion protein according to claim 3, characterized in that: In step (5), the specific steps of fermentation and purification are: The monoclone grown on the bleomycin plate was picked up, inoculated into a conical flask containing BMGY medium, and cultured overnight as a seed solution; the seed solution was inoculated into a conical flask containing BMGY medium, cultured to the logarithmic growth phase, and methanol was added for induction; thereafter, methanol was regularly added to the culture medium to maintain continuous expression of the induction; after the induction expression was completed, the supernatant was retained by centrifugation, and the supernatant was subjected to protein precipitation, centrifugation, suspension, and dialysis to obtain the target protein.

5. Use of the anticoccidial fusion protein according to claim 1 or 2 in the preparation of anticoccidial insecticides.

6. The use according to claim 5, characterized in that: The anti-coccidial fusion protein uses LiPM to enzymatically hydrolyze the outer layer of the coccidia oocyst wall, and APP enters the inner layer of the eggs to eliminate the eggs and exert its effect.

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