Anticococcal fusion protein and its preparation method and application

Abstract

The present invention relates to the fields of gene recombination technology and pharmaceutical preparations, and provides an anticoccidial fusion protein, the amino acid sequence of which is composed of a broad-spectrum antiprotozoal 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 antiprotozoal 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. The present invention also provides a preparation method and application of the above-mentioned anticoccidial fusion protein. The present invention is designed based on APP and LiPM. When used as an insecticide, the outer layer of the oocyst wall can be enzymatically decomposed by LiPM, while APP can enter the inner layer of the eggs to effectively kill the eggs, thereby achieving a better anticoccidial effect.

Description

Technical Field

[0001] The present invention relates to the fields of gene recombination technology and pharmaceuticals, and in particular to an anticoccidial fusion protein and a preparation method and application thereof. Background Art

[0002] Our prior patent, CN 116903753B, discloses a broad-spectrum antiprotozoal peptide. The peptide's amino acid sequence is composed of a linked sequence of HIV-TAT transduction protein, a flexible linker protein, and a mutant APLV4-VP protein. This peptide demonstrates broad-spectrum antiprotozoal efficacy comparable to antibiotics without inducing drug resistance, providing a safe and effective non-antibiotic antiprotozoal drug for the aquaculture industry. However, farmers have reported that this drug is only effective against protozoa other than coccidia, and that coccidia infections are prone to relapse after treatment.

[0003] The HIV-TAT transduction protein is a small molecule protein that can cross host cell membranes through its unique cell-penetrating peptide (CPP) structure. It typically interacts with lipids on the cell membrane or guides the proteins it carries into the cell through endocytosis, where they play a vital role. The coccidian oocyst is a key transmission structure in the coccidian life cycle. It consists of two layers, the inner and outer layers, each with distinct functions and compositions. The inner layer, similar to a normal cell membrane, is primarily composed of a complex of proteins, lipids, and polysaccharides. The outer layer is composed of collagen-like proteins and keratin cross-linked by tyrosine, forming a robust three-dimensional network. This prevents the HIV-TAT transduction protein from transporting the mutant APLV4-VP protein through the outer oocyst membrane and into the eggs, where it can kill them. However, in the middle and late stages of coccidian infection, eggs have already begun to form. Therefore, the approach described in patent CN 116903753B is not yet effective in killing these eggs in vivo. Once the eggs are expelled from the body, if medication is discontinued, ingestion of the eggs can lead to reinfection within the colony.

[0004] Fusion proteins are created through genetic recombination techniques by precisely linking specific functional regions or complete proteins from different proteins at the amino acid level, creating novel proteins with multiple biological activities. This fusion strategy achieves the synergistic integration of different protein functional domains, enabling recombinant fusion proteins to exhibit superior biological properties compared to single proteins. Recombinant fusion proteins demonstrate enormous potential and value in scientific fields such as medicine, biotechnology, and vaccine development, for example, in the development of novel therapeutics for anti-tumor, anti-rheumatic, and autoimmune diseases.

[0005] Based on this, the present invention attempts to combine fusion protein technology to develop a drug with better anticoccidial effect, especially a better killing effect on coccidian eggs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an anticoccidial fusion protein and its preparation method and application. The anticoccidial fusion protein is designed based on a broad-spectrum antiprotozoan polypeptide (APP) and a lignin peroxidase mutant (LiPM). When used as an insecticide, the outer layer of the oocyst wall can be enzymatically degraded by LiPM, while APP can penetrate the inner layer of the eggs to effectively kill the eggs, thereby achieving a better anticoccidial effect.

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

[0008] Disclosed is an anticoccidial fusion protein, wherein the amino acid sequence of the anticoccidial fusion protein is composed of a broad-spectrum antiprotozoal 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 antiprotozoal 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.

[0009] As one of the preferred embodiments of the present invention, the lignin peroxidase mutant LiPM is specifically derived from 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.

[0010] A method for preparing the above-mentioned anticoccidial fusion protein comprises 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) APP protein and LiPM protein were linked using an oxidative stress-sensitive linker and a flexible linker to obtain the amino acid sequence of the anti-coccidial fusion protein, as shown in SEQ ID NO.6;

[0013] (3) Using DNAMAN software and combined with Saccharomyces cerevisiae codon preference optimization, the amino acid sequence of the anti-coccidial 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-coccidial fusion protein, as shown in SEQ ID NO.7;

[0014] (4) Double-digest the fusion protein gene sequence and expression plasmid using endonucleases, ligate the digested products, and transform them into cloning bacteria; screen positive clones, extract the plasmids, and perform sequencing verification; linearize the verified plasmids and then introduce them into the expression bacteria; select high-copy transformants and culture them to obtain the target yeast engineering bacteria;

[0015] (5) Fermentation and purification of anti-coccidian fusion protein.

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

[0017] A single clone growing on a bleomycin plate was picked and 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 and cultured to the logarithmic growth phase, and methanol was added for induction; thereafter, methanol was regularly added to the culture medium to maintain continuous induced expression; after the induction expression was completed, the supernatant was centrifuged and retained, and the supernatant was subjected to protein precipitation, centrifugation, suspension, and dialysis to obtain the target protein.

[0018] A use of the anticoccidial fusion protein in the preparation of anticoccidial insecticides.

[0019] As one of the preferred embodiments of the present invention, the anti-coccidian fusion protein degrades the outer layer of the coccidian oocyst wall through LiPM, and APP enters the inner layer of the eggs to eliminate the eggs and exert its effect.

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

[0021] Lignin peroxidase (LiP) can enzymatically degrade lignin, mainly recognizing benzene ring structures and destroying CC bonds. The wall of the oocyst of coccidian eggs is mainly cross-linked by tyrosine to form a dityrosine bond that is difficult to be degraded by digestive enzymes such as pancreatic enzymes. It has a benzene ring structure that can be recognized by LiP in structure, and the dityrosine bond is CC, so lignin peroxidase has the possibility of degrading the dityrosine bond. However, in order to make lignin peroxidase better degrade the dityrosine bond, the binding center of LiP - the heme binding residue site can be mutated. Specifically, the present invention selects the mature peptide sequence of the fungus LiP (NCBI accession number AAA34049, the amino acid sequence is shown in SEQ ID NO.5), and mutates its 179th H to A, and the 180th T to F. The amino acid sequence after mutation is shown in SEQ ID NO.3, which is recorded as the lignin peroxidase mutant LiPM.

[0022] Based on this, applying LiPM to the wall breaking of coccidian oocysts can effectively solve the problem that coccidian eggs cannot be broken by enzymatic hydrolysis under conventional conditions. Further combined with the broad-spectrum antiprotozoal polypeptide (denoted as APP) disclosed in our company's prior patent CN 116903753B, APP can enter the inner layer of the eggs and effectively kill the eggs.

[0023] The advantages of the present invention over the prior art are:

[0024] (1) The present invention is based on the design and preparation of a fusion protein based on the broad-spectrum antiprotozoal polypeptide APP and the lignin peroxidase mutant LiPM; when used as an insecticide, the fusion protein LiPM can effectively enzymatically degrade the outer layer of the oocyst wall, and then APP can enter the inner layer of the egg and effectively kill the egg, achieving a better anticoccidial effect;

[0025] (2) The fusion protein of the present invention uses an oxidative stress-sensitive linker and a flexible linker as connecting peptides, wherein the flexible linker can separate APP and LiPM in space without affecting their respective biological functions; while the oxidative stress-sensitive linker breaks when LiPM enzymatically degrades the dityrosine bond, allowing APP to separate, thereby entering the inner layer of the oocyst more effectively and quickly;

[0026] (3) The present invention adopts Pichia pastoris fermentation expression, which has a higher level of structure and better activity than prokaryotic expression, and the product post-processing process is simple, low-cost, and can be widely used. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This 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 These are microscopic results of the enzymatic hydrolysis of coccidia eggs by FACP and APP-LiP of the present invention (Figure A is a microscopic examination picture after enzymatic hydrolysis of APP-LiP; Figure B is a microscopic examination picture after enzymatic hydrolysis of FACP; 400×). DETAILED DESCRIPTION

[0029] The following examples of the present invention are described in detail. These examples are based on the technical solutions of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are conventional materials purchased directly from existing biochemical reagent stores unless otherwise specified.

[0030] Example 1. Design of anticoccidial fusion protein:

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

[0032] The lignin peroxidase mutant (LiPM) is derived from the lignin peroxidase (LiP) shown in 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.

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

[0034] Example 2: Obtaining the anticoccidial fusion protein nucleic acid sequence:

[0035] Using DNAMAN software and combined with Saccharomyces cerevisiae codon preference optimization, the amino acid sequence of the anticoccidial 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 anticoccidial fusion protein, as shown in SEQ ID NO.7.

[0036] Example 3, Preparation of anticoccidial fusion protein engineered bacteria:

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

[0038] Use chemical transformation to transfer the FACP-pT19 plasmid into DH5α clones, plate onto ampicillin-resistant LB plates, and culture overnight. Select positive clones and incubate them in ampicillin-resistant LB liquid medium at 37°C, 220 rpm, for 8 hours. Store the remaining culture in a culture 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 double-digested directly with EcoRⅠ and NotⅠ endonucleases, respectively.

[0040] The target gene segment after pT19 digestion and the pPiczαA backbone segment were recovered by gel excision. The two recovered products were ligated using Takara's T4 ligase and then chemically transformed into DH5α cloning bacteria.

[0041] Screen positive clones using LB plates containing 25 μg / mL bleomycin. Select positive clones and incubate them in LB liquid medium containing 25 μg / mL bleomycin at 37°C, 220 rpm, for 8 hours. Store the clones as seed stock and continue incubating the remaining culture overnight. Extract the plasmids and send them to BGI for sequencing verification.

[0042] The verified plasmid was linearized using SacⅠ endonuclease.

[0043] Take OD 600 Centrifuge X33 yeast cells at 4000 rpm for 10 minutes after reaching a density of 0.6 to 1.0. Discard the supernatant and wash the cells twice with ice-cold 1M LiAc to remove salt from the culture medium. Resuspend the washed yeast cells in 1M LiCl and perform electroporation using the following conditions: voltage: 2.0 kV / cm; pulse width: 5 ms; capacitance: 100 μF; conductivity: 800 Ω.

[0044] After electroporation, cells were transferred to liquid culture medium containing 1M SOS and incubated at 30°C for 1 hour. The pellet was then centrifuged at 4000 rpm for 10 minutes and resuspended in 200 μL of sterile water. The pellet was then plated onto YPDS plates containing 1 mg / mL bleomycin and incubated inverted in a 30°C incubator for 5 days. Positive transformants were selected and cultured at a constant temperature to obtain the target engineered yeast strain, designated FACP-X33.

[0045] Example 4: Fermentation and purification of anticoccidial fusion protein

[0046] Take FACP-X33 and draw four lines on a YPDS plate containing 1 mg / mL bleomycin. Place the plate upside down in a 30°C incubator and incubate for 5 days. Pick the positive transformants and inoculate them into a 50ml conical flask containing 20ml BMGY medium. Incubate at 30°C, 220 rpm overnight until the OD reaches 0. 600 =4, as seed solution, inoculated into a 2L conical flask containing 1L BMGY medium, and cultured at 30℃, 250r / min for about 20h until OD 600 =30, add 0.5% v / v methanol for induction, set the temperature to 28°C, and the rotation speed to 220 rpm. Thereafter, add 0.5% methanol to the culture medium every 24 hours to maintain continuous induced expression. Induction expression is stopped for 48 hours.

[0047] Centrifuge at room temperature, 4000r / min for 10 minutes and retain the supernatant. Take the supernatant after centrifugation and add saturated ammonium sulfate in a 1:1 ratio. Then place the mixture in a 4°C refrigerator for 24 hours to allow the protein in the fermentation broth to flocculate and precipitate. Centrifuge at 4°C, 12000rpm for 15 minutes, take the precipitate, and discard the supernatant. Suspend the precipitate with an appropriate amount of PBS buffer and dialyze with PBS of the same concentration as the suspension to remove salts to obtain the target protein FACP. Figure 1 Lane 2 shows.

[0048] Comparative Example 1: Preparation of anticoccidial fusion protein without mutation of LiP:

[0049] An unmutated LiP anticoccidial fusion protein was designed, with the amino acid sequence of the protein being: 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 resulting unmutated LiP anticoccidial fusion protein has an amino acid sequence as shown in SEQ ID NO. 8, and the corresponding nucleic acid sequence obtained according to Example 2 is shown in SEQ ID NO. 9.

[0050] Further, referring to the methods of Examples 3 and 4 above, the designed "LiP non-mutated anticoccidial fusion protein" was constructed by engineering bacteria and the protein was expressed and purified to obtain the corresponding target protein, which was recorded as APP-LiP. Figure 1 Lane 3 shows.

[0051] Experimental Example 1: Enzymatic hydrolysis of coccidia eggs:

[0052] Simulating the pH of the chicken small intestine cecum, the anticoccidial fusion protein (FACP) of the present invention and the anticoccidial fusion protein (APP-LiP) of the unmutated lignin peroxidase of Comparative Example 1 were dissolved in PBS at pH 6.0 to a concentration of 0.1 mg / mL. 10 mL of each dissolved protein solution was added to 1 mL of 10,000 coccidian eggs / mL, resulting in a final egg concentration of approximately 1,000 eggs / mL. Three replicates were performed.

[0053] Simulating the chicken's body temperature and digestive tract transit time, place the eggs in a 41°C constant temperature water bath shaker and incubate for 4 hours. Observe and count the intact eggs under a microscope.

[0054] The results are shown in Table 1 and Figure 2 shown. Figure 2 In Figure A, the eggs are still intact and the membrane structure is clearly visible. In Figure B, the oocysts are ruptured and sporozoites are released.

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

[0056] Group FACP APP-LiP The average number of intact eggs 4526 10000 The proportion of membrane-broken eggs 54.7％ 0％

[0057] From Table 1 and Figure 2 The results showed that APP-LiP had no effect on enzymatically hydrolyzing worm eggs or breaking the membrane after enzymatically hydrolyzing worm eggs, while FACP had a certain effect on enzymatically hydrolyzing worm eggs.

[0058] Experimental Example 2: Verification of the anticoccidial effect of anticoccidial fusion protein:

[0059] (1) Drug treatment

[0060] After the anticoccidial fusion protein (FACP) is freeze-dried, the test dose is 100 grams per ton of feed; after the broad-spectrum antiprotozoal polypeptide (APP) is freeze-dried, the test dose is 100 grams per ton of feed.

[0061] Use PBS as the diluent for spraying FACP to prepare a 10% FACP protein solution, add 10% glycerol to maintain wettability, add 2% saponin to improve uniformity, mix well and store at 4°C for later use.

[0062] (2) Coccidia attack and treatment

[0063] The experimental animals were 30-day-old white-feathered chickens free of Eimeria infection. Groups were divided according to Table 2: 24 chickens in each group (except for the 7-day FACP treatment group), and 36 chickens in each standard group. Each group was housed in a coccidian-free environment. Each chicken was orally administered 10,000 Eimeria eggs. Daily observation and record keeping of water intake, food intake, feces, and mortality were performed for each group. Dead chickens were promptly dissected for cause of death analysis. Four days after treatment, medicated feed was initiated. Daily observation and record keeping of water intake, food intake, feces, and mortality were performed for each group. Dead chickens were promptly dissected for cause of death analysis. After three days of treatment, medication was discontinued, and 10 surviving chickens from each group were sacrificed by dislocation and weighed. The cecum was removed, and the mucosa and contents were scraped and the number of oocysts per chicken was determined. Feeding was continued for another 5 days using a blank, unmedicated diet in the original environment. Ten surviving chickens from each group were sacrificed by dislocation and weighed. The cecum was removed, the cecal mucosa and contents were scraped, and the number of oocysts in each chicken was detected and calculated. The feed supplemented with drugs was continued to be fed in the original environment for 10 days. Ten surviving chickens in each group were killed by dislocation of the neck. Weigh them. The cecum was removed, the cecal mucosa and contents were scraped, and the number of oocysts in each chicken was detected and calculated. The FACP continuous treatment group (including 7 days of continuous treatment with FACP for parasite attack + FACP spraying group) continued to use FACP treatment for 7 days after 4 days of parasite attack, then stopped the drug and continued feeding with blank feed. After 7 days of treatment, 10 surviving chickens were killed by dislocation of the neck. Weigh them. The cecum was removed, the cecal mucosa and contents were scraped, and the number of oocysts in each chicken was detected and calculated. And after 8 days, 10 surviving chickens were killed by dislocation of the neck together with the other groups. Weigh them. The cecum was removed, the cecal mucosa and contents were scraped, and the number of oocysts in each chicken was detected and calculated. The FACP spraying method for disinfecting the environment was to disinfect twice a day until the end of the experiment for the corresponding group.

[0064] Table 2 Coccidia attack and treatment groups

[0065]

[0066]

[0067] (3) Evaluation of anticoccidial effect

[0068] The anticoccidial index (ACI) was calculated as follows: ACI = (survival rate + relative weight gain) - (lesion value + oocyst value); survival rate = (number of surviving chickens at the end of the test / number of test chickens) × 100%; relative weight gain = (weight gain of the test group / weight gain of the blank control group) × 100%. An ACI ≥ 160 was considered excellent, an ACI of 130-159 was considered good, an ACI of 100-129 was considered hyposensitive, and an ACI < 100 was considered ineffective. The ACI results for each group are shown in Table 3.

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

[0070]

[0071]

[0072] The dose used to treat each chicken in this experiment was relatively low, but the drug was used starting four days after treatment, at which point the first generation of worm eggs had already begun to form in the chickens of each treated group. From the perspective of the antiprotozoal index (ACI), the ACI values for groups 6 to 9 after seven consecutive days of FACP use were 147.1, 170.4, 171.7, and 187.4, all showing good or above results. In particular, groups 8 and 9 showed no reinfection after seven consecutive days of use and eight days of drug withdrawal, with ACI values of 200, demonstrating outstanding results. Comparison of groups treated with FACP spraying and those not treated with FACP spraying suggests that FACP spraying may have a certain killing effect on worm eggs in the field, thereby helping the various drugs to achieve better results. Accordingly, the present invention provides a fusion protein with better anticoccidial effects than a "broad-spectrum antiprotozoal polypeptide."

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection 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 antiprotozoal 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 antiprotozoal 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, wherein The lignin peroxidase mutant LiPM is specifically derived from 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 the 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) APP protein and LiPM protein were linked using an oxidative stress-sensitive linker and a flexible linker to obtain the amino acid sequence of the anti-coccidian fusion protein, as shown in SEQ ID NO.6; (3) Using DNAMAN software and 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 Not Ⅰ were added downstream to obtain the nucleic acid sequence of the anti-coccidial fusion protein; (4) Double-digest the nucleic acid sequence of the fusion protein and the expression plasmid using endonucleases, ligate the digested products, and transform them into cloning bacteria; screen positive clones, extract the plasmids, and perform sequencing verification; linearize the verified plasmids and then introduce them into yeast; select high-copy transformants for culture to obtain the target yeast engineered bacteria; (5) Fermentation and purification of anti-coccidian fusion protein.

4. The method for preparing anticoccidial fusion protein according to claim 3, wherein: In step (5), the specific steps of fermentation and purification are: A single clone growing on a bleomycin plate was picked and 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 and cultured to the logarithmic growth phase, and methanol was added for induction; thereafter, methanol was regularly added to the culture medium to maintain continuous induced expression; after the induction expression was completed, the supernatant was centrifuged and retained, 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 an anti-Eimeria insecticide.

6. The use according to claim 5, characterized in that The anti-coccidial fusion protein uses LiPM to enzymatically decompose the outer layer of the coccidian oocyst wall, and the broad-spectrum antiprotozoal polypeptide APP enters the inner layer of the eggs to eliminate the eggs and exert its effect.

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