Broad-spectrum antiprotozoal polypeptide and preparation and use thereof

Abstract

Provided is a broad-spectrum antiprotozoal polypeptide, an amino acid sequence of which is formed by sequentially connecting a transduction protein domain sequence, a linker protein sequence, and a mutant APLV4-VP protein sequence, wherein the transduction protein domain sequence is an HIV-TAT transduction protein domain sequence, the linker protein sequence is a flexible linker protein sequence, and the mutant APLV4-VP protein sequence comprises a mutant VP4 protein and original VP2, VP3 and VP1 protein sequences. Also provided are preparation of the polypeptide and an antiprotozoal use of the polypeptide in vivo and / or in vitro in animals. On the basis of a mutant APLV4-VP protein, the feasibility of transmembrane transduction of the mutant APLV4-VP protein is realized by means of a transduction protein domain, so that an artificial antiprotozoal polypeptide is designed; the polypeptide can be used for broad-spectrum antiprotozoal, has the effect not inferior to that of antibiotic drugs, and would not cause drug resistance.

Description

A broad-spectrum antiprotozoal polypeptide and its preparation and application Technical Field

[0001] The present invention relates to the fields of gene recombination technology and pharmaceutical technology, and in particular to a broad-spectrum antiprotozoal polypeptide and its preparation and application. Background Art

[0002] Recombinant proteins are proteins obtained using recombinant DNA or recombinant RNA technology. Recombinant protein engineering begins by obtaining the target gene through gene cloning or chemical synthesis, connecting it to a suitable expression vector, and introducing it into a specific host cell. The host cell's genetic system is then utilized to express a functional protein molecule.

[0003] Cell-penetrating peptides (CPPs) are a class of short peptides capable of crossing cell membranes or tissue barriers. They can transport biomacromolecules such as proteins, RNA, and DNA into cells through mechanisms such as endocytosis and direct penetration, where they can exert their effector functions. Compared to other non-natural chemical molecules, CPPs offer advantages such as excellent biocompatibility, minimal cellular toxicity, and the ability to be directly fused with biologically active proteins for recombinant expression. Many CPPs are derived from protein transduction domains of viral structural proteins that directly interact with host cells.

[0004] Apis picorna-like virus 4 (APLV4) is a positive-strand RNA virus that is transmitted by honeybees and varroa mites. It is prevalent and persistent in Chinese bee colonies. Infection with the virus causes significant mortality in adults, larvae, and pupae. In addition to worker bee, larvae, and pupae mortality, queen bees are also directly or indirectly affected. In the later stages of APLV4 infection, queen weight and egg production decrease. The virus also causes significant mortality in eggs, nymphs, and adults of varroa mites.

[0005] Protozoa are heterotrophic, single-celled eukaryotic microorganisms. Their basic structure consists of a membrane, cytoplasm, and nucleus. They range in size from 1 to 150 μm and possess a simple structure. However, during their long evolutionary adaptation to a parasitic life, they have undergone functional differentiation at the cytoplasmic level, developing specialized organelles that perform functions similar to those of organs in higher animals. Since Leeuwenhoek first discovered Eimeria steussenii in the 17th century, over 200,000 species of protozoa have been documented. Protozoa are ubiquitous, found in polar soils and waters. Over 10,000 known species of protozoa are parasitic, and some are important pathogens, causing significant diseases in both animals and humans, such as malaria, coccidiosis, piroplasmosis, and trichomoniasis, causing significant harm to human health and economic development. Current treatment options primarily rely on nidazole antibiotics. With the implementation of antimicrobial bans, the overuse of antibiotics is becoming increasingly concerning, as it can lead to the emergence of drug resistance in protozoa. For breeding companies, the occurrence of protozoal diseases in products before they are released to the market is a serious blow. Therefore, a safe and effective non-antibiotic antiprotozoal drug has become an urgent need for many breeding companies.

[0006] Currently, patent CN101048154A discloses an antiprotozoal agent, but this agent is only effective against Leishmania and lacks a broad-spectrum antiprotozoal agent. It is therefore unsuitable for use in mixed infections, a prominent problem in domestic aquaculture. Therefore, the method disclosed in CN101048154A still cannot meet the current needs of the domestic aquaculture industry.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a broad-spectrum antiprotozoal polypeptide and its preparation and application. The polypeptide is designed based on the mutant APLV4-VP protein and the transduction protein domain (as a cell-penetrating peptide) to design an artificial antiprotozoal polypeptide. The polypeptide can be used for broad-spectrum antiprotozoal activities, and its effect is no less than that of antibiotics without causing drug resistance.

[0009] The present invention adopts the following technical solutions to solve the above technical problems:

[0010] A broad-spectrum antiprotozoal polypeptide, wherein the amino acid sequence of the broad-spectrum antiprotozoal polypeptide is composed of a transduction protein domain sequence, a linker protein sequence, and a mutant APLV4-VP protein sequence connected in sequence; wherein the transduction protein domain sequence is the HIV-TAT transduction protein domain amino acid sequence; and the linker protein sequence is a flexible linker protein amino acid sequence (GGGGS). n , n≥1; the mutant APLV4-VP protein sequence includes a mutant VP4 protein amino acid sequence and original VP2, VP3, and VP1 protein amino acid sequences connected sequentially.

[0011] As one of the preferred embodiments of the present invention, the amino acid sequence of the HIV-TAT transduction protein domain is shown as SEQ ID NO.1; the amino acid sequence of the flexible linker protein is shown as SEQ ID NO.2; in the mutant APLV4-VP protein, the amino acid sequence of the mutant VP4 protein is shown as SEQ ID NO.3, the amino acid sequences of the unmutated original VP2, VP3, and VP1 proteins are shown as SEQ ID NO.4, 5, and 6, respectively, and the complete amino acid sequence of the mutant APLV4-VP protein is shown as SEQ ID NO.7.

[0012] A method for preparing the above-mentioned broad-spectrum antiprotozoal polypeptide comprises the following steps:

[0013] (1) Obtaining the gene sequence of broad-spectrum antiprotozoal polypeptides;

[0014] (2) Construction of engineered bacteria;

[0015] (3) Fermentation and purification to obtain broad-spectrum antiprotozoal peptides.

[0016] As one of the preferred embodiments of the present invention, the broad-spectrum antiprotozoal polypeptide gene sequence in step (1) is obtained by:

[0017] ① Obtain the transduction protein domain sequence;

[0018] ② Obtain the mutant APLV4-VP protein sequence;

[0019] ③ Using a linker protein sequence to connect the transduction protein domain sequence and the mutant APLV4-VP protein sequence to obtain an amino acid sequence of a broad-spectrum antiprotozoal polypeptide;

[0020] ④ Use DNAMAN software for reverse translation, optimize it based on the codon preference of Bacillus subtilis, add the restriction site BamHⅠ upstream and the restriction site XbaⅠ downstream to obtain the gene sequence of the broad-spectrum antiprotozoal polypeptide.

[0021] As one of the preferred embodiments of the present invention, the engineering bacteria in step (2) are constructed by performing double enzyme digestion on the obtained gene sequence of the broad-spectrum antiprotozoal polypeptide and the pHT43 plasmid using an endonuclease, connecting the double enzyme digestion products using T4 ligase, picking positive single colonies, expanding and culturing at a constant temperature, and sending them for detection to obtain qualified ones.

[0022] As one of the preferred embodiments of the present invention, the fermentation purification in step (3) is carried out by the following specific fermentation purification method:

[0023] ① Take the qualified engineered bacteria after sequencing and culture them in a shaker at 37°C and 220 rpm until OD600 reaches 2.0; add 3mM IPTG, then adjust the shaker temperature to 18°C, and harvest after induction for 12 hours; centrifuge at 4°C and 12000 rpm for 15 minutes, remove the supernatant and discard the precipitate;

[0024] ② Take the supernatant after centrifugation and add 80% saturation (NH4)2SO4 in a 1:1 ratio; then place the mixture in a 4°C refrigerator and let it stand for 48 hours to flocculate and precipitate the protein in the fermentation broth; centrifuge at 4°C and 12000rpm for 15 minutes, take the precipitate, and discard the supernatant; suspend the precipitate with PBS buffer and dialyze with PBS of the same concentration as the suspension to remove salt; take the dialyzed protein solution and add 80% saturation (NH4)2SO4 in a 1:1 ratio; then place the mixture in a 4°C refrigerator and let it stand for 48 hours to flocculate and precipitate the protein in the mixture; centrifuge at 4°C and 12000rpm for 15 minutes, take the precipitate, and discard the supernatant; suspend the precipitate with PBS buffer and dialyze with PBS of the same concentration as the suspension to remove salt to obtain the target protein.

[0025] A use of the broad-spectrum antiprotozoal polypeptide in animals in vivo and / or in vitro.

[0026] As one of the preferred embodiments of the present invention, when an animal is orally administered with the broad-spectrum antiprotozoal polypeptide, the broad-spectrum antiprotozoal polypeptide exerts a broad-spectrum antiprotozoal effect in the animal.

[0027] As one of the preferred embodiments of the present invention, after the broad-spectrum antiprotozoal polypeptide is sprayed in the animal breeding environment, the broad-spectrum antiprotozoal polypeptide exerts a broad-spectrum antiprotozoal effect outside the animal's body.

[0028] The advantages of the present invention over the prior art are:

[0029] (1) The present invention is designed based on the mutant APLV4-VP protein, and the feasibility of transmembrane transduction of the mutant APLV4-VP protein is achieved by pre-positioning the transduction protein domain, thereby designing an artificial antiprotozoal polypeptide (the transduction protein domain acts as a cell-penetrating peptide, guiding the APLV4-VP protein into cells, thereby improving the antiprotozoal effect); the polypeptide can be used for broad-spectrum antiprotozoal effects, with an effect comparable to that of antibiotics and without causing drug resistance;

[0030] (2) The present invention adopts Bacillus subtilis fermentation expression, and the post-processing process of the product is simple and low-cost, and can be widely used in various types of breeding industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a graph showing the CPE of the bee tissue grinding solution in Example 2 after 17 consecutive generations of proliferation of Vero cells ( FIG1 , FIG1 A shows the CPE of the bee tissue, and FIG1 B shows the Vero cell control);

[0032] FIG2 is a diagram showing the comparison of the sequencing results of the mutant APLV4-VP protein and the unmutated VP sequence in Example 2;

[0033] FIG3 is a Page electrophoresis diagram of the broad-spectrum antiprotozoal polypeptide in Example 3 (in FIG3 , M: protein Marker 26610; lane 1: recombinant broad-spectrum antiprotozoal polypeptide; lane 2: empty bacteria control);

[0034] FIG4 is a diagram showing the entire cell growth process derived by the RTCA SP Instrument system in Example 4 (in FIG4 , the ordinate represents the cell growth index CGI, and the abscissa represents the culture time). DETAILED DESCRIPTION

[0035] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are conventional materials directly purchased from existing biochemical reagent stores unless otherwise specified.

[0037] Example 1: Broad-spectrum antiprotozoal polypeptides

[0038] The amino acid sequence of the broad-spectrum antiprotozoal polypeptide is composed of a transduction protein domain sequence, a linker protein sequence and a mutant APLV4-VP protein sequence connected in sequence.

[0039] The transduction protein domain sequence is the HIV-TAT transduction protein domain amino acid sequence, as shown in SEQ ID NO.1.

[0040] The linker protein sequence is a flexible linker protein amino acid sequence (GGGGS) n , n≥1, as shown in SEQ ID NO.2.

[0041] The mutant APLV4-VP protein sequence comprises a sequentially linked mutant VP4 protein amino acid sequence and the original unmutated VP2, VP3, and VP1 protein amino acid sequences. The mutant VP4 protein amino acid sequence is shown in SEQ ID NO. 3, the original unmutated VP2, VP3, and VP1 protein amino acid sequences are shown in SEQ ID NOs. 4, 5, and 6, respectively, and the complete mutant APLV4-VP protein amino acid sequence is shown in SEQ ID NO. 7.

[0042] Example 2: Obtaining the mutant APLV4-VP protein sequence

[0043] While developing a treatment for the fever mite (Varella mites), our company discovered that one bee colony in a test apiary showed no cysticerosis infection under microscopic examination, while all other bee colonies in the test apiary showed cysticerosis. Experiments ruled out the drug's efficacy against the Varella mites. 16S rRNA RT-PCR amplification and sequencing ruled out pathogenic bacteria.

[0044] Several tissues of bees from the colony were ground and inoculated into Vero cells. After 17 generations of continuous proliferation in a serum-free medium containing 150ug / mL trypsin, cytopathic effect (CPE) was obvious (as shown in Figure 1, CPE was syncytial and fragmented). The cells were frozen and thawed three times and centrifuged at 12000rpm to remove cell debris. The cells were then purified using sucrose density gradient centrifugation. RT-PCR and PCR were used to detect and exclude 12 common viral infections such as wing-deleted virus and Sinai Lake virus. The purified samples were sent to BGI and Sangon Biotech for whole-genome sequencing and N-terminal sequencing, respectively. BLAST revealed that the cells were highly homologous to the existing Apis picorna-like virus 4 but had partial mutations in VP 4 (sequence alignment results are shown in Figure 2). The structural protein amino acid sequence: VP4 (mutation)-VP2-VP3-VP1 is shown in SEQ ID NO.7 and is recorded as mutant APLV4-VP protein.

[0045] Example 3: Preparation of broad-spectrum antiprotozoal polypeptides

[0046] (1) Obtaining the TAT-VP fusion protein gene sequence

[0047] With reference to NCBI accession number 8CCZ_C, the HIV-TAT sequence was obtained to obtain the amino acid sequence of the HIV-TAT transduction protein domain, as shown in SEQ ID NO.1.

[0048] The amino acid sequence of the mutant APLV4-VP protein was obtained according to the method in Example 2, as shown in SEQ ID NO.7.

[0049] The linker protein shown in SEQ ID NO.2 was used to connect the HIV-TAT transduction protein domain sequence and the amino acid sequence of the mutant APLV4-VP protein to obtain the target protein amino acid sequence shown in SEQ ID NO.8.

[0050] DNAMAN software was used for reverse translation and optimization based on the codon preference of Bacillus subtilis. The restriction site BamHⅠ and the start codon were added upstream, and the stop codon and the restriction site XbaⅠ were added downstream to obtain an artificial nucleic acid sequence of a broad-spectrum antiprotozoal polypeptide, as shown in SEQ ID NO.9. The gene sequence was sent to BGI for synthesis, and after sequencing, the nucleic acid sequence of the target protein was obtained.

[0051] (2) Construction of engineered bacteria

[0052] The synthesized target gene was double-digested with BamHⅠ and XbaⅠ endonucleases on the synthesized gene and pHT43 plasmid; the double-digested products were ligated using ligase and introduced into WB800N competent cells; the cells were spread on ampicillin LB plates and cultured at a constant temperature of 37°C; single colonies grown on the ampicillin LB plates were picked for expansion and sampling and sent to BGI for sequencing; the sequencing results were consistent with the target gene, indicating that the expression bacteria were successfully constructed and recorded as TAT-VP engineered bacteria.

[0053] (3) Fermentation and purification

[0054] The qualified TAT-VP engineered bacteria after sequencing were cultured using LB medium on a shaker at 37°C and 220rpm until OD600 was 2.0; IPTG was added at a final concentration of 3mM, and the shaker temperature was adjusted to 18°C. After induction for about 12 hours, the mixture was harvested; the mixture was centrifuged at 4°C and 12000rpm for 15 minutes, the supernatant was taken and the precipitate was discarded; the supernatant after centrifugation was added with 80% saturation (NH4)2SO4 in a 1:1 ratio; the mixture was then placed in a 4°C refrigerator for 48 hours to allow the protein in the fermentation broth to flocculate and precipitate; the mixture was placed in a 4°C refrigerator for 12000rpm and the mixture was centrifuged at 4°C and 12000rpm for 15 minutes. pm centrifuged for 15 minutes, took the precipitate, and discarded the supernatant; suspended the precipitate with PBS buffer, and dialyzed with PBS of the same concentration as the suspension to remove salt; took the dialyzed protein solution and added 80% saturation (NH4)2SO4 in a 1:1 ratio; then placed the mixture in a 4°C refrigerator for 48 hours to allow the protein in the mixture to flocculate and precipitate; centrifuged at 4°C, 12000 rpm for 15 minutes, took the precipitate, and discarded the supernatant; suspended the precipitate with PBS buffer, and dialyzed with PBS of the same concentration as the suspension to remove salt.

[0055] Preliminary SDS-Page analysis revealed that the target protein was the expected size (18 kD, as shown in Figure 3). The resulting protein was identified as the target protein—a broad-spectrum antiprotozoal polypeptide, designated as Anti-protozoon polypeptide, APP.

[0056] Example 4: Verification of the Antiprotozoal Effect of Broad-spectrum Antiprotozoal Peptides in Vitro

[0057] (1) Parameter settings

[0058] Create a new experiment on the RTCA SP Instrument system and set:

[0059] Step 1: 1; 1min; baseline measurement, fixed detection 1min once;

[0060] Step 2: 36; 30min; 18hrs, used to detect cell proliferation process;

[0061] Step 3: 144; 30min; 72hrs, used to detect protozoan attack and drug effects;

[0062] (2) Cell culture

[0063] Vero cells were collected at 1.5×10 4 The cells were inoculated into Agilent RTCA E-Plate 96-well cell plates and cultured on the RTCA SP Instrument system in a 5% CO2 incubator for 24 hours before the insect attack experiment.

[0064] (3) Attacking insects and evaluating them

[0065] On a 96-well E-Plate, 32 wells were challenged with Eimeria at 100 cells / well, 16 wells with Plasmodium at 100 cells / well, and 16 wells with Trichomonas at 100 cells / well. The remaining 32 wells were not challenged with any parasites, and diluent (2% CS in DMEM) was added.

[0066] At the same time, in the group attacking Eimeria, 2ug of TAT-Linker polypeptide (HIV-TAT transduction protein domain + flexible linker protein), 2ug of VP polypeptide (mutant APLV4-VP protein) were added to 8 wells, 2ug of the target protein of the present invention (HIV-TAT transduction protein domain + flexible linker protein + mutant APLV4-VP protein, APP) were added to 8 wells, and diluent was added to 8 wells. In the group attacking Plasmodium, 2ug of the target protein of the present invention (APP) was added to 8 wells, and diluent was added to 8 wells. In the group attacking Trichomonas, 2ug of the target protein of the present invention (APP) was added to 8 wells, and diluent was added to 8 wells. In the group not attacking protozoa, 20ug of TAT-Linker polypeptide, 2ug of VP polypeptide, 2ug of APP were added to 8 wells, and diluent was added to 8 wells. Culture was carried out on the RTCA SP Instrument system in a 5% CO2 incubator for 72 hours.

[0067] The cell growth curve on the RTCA SP Instrument system was derived, as shown in Figure 4, where the ordinate represents the cell growth index (CGI), which is the fitted value of cell viability and cell number, and the abscissa represents the culture time.

[0068] As shown in Figure 4, the parasite-treated group treated with the target protein of the present invention (broad-spectrum antiprotozoal polypeptide, APP) showed a slightly lower CGI than the blank control and negative control groups, but exhibited significantly better growth and proliferation activity than all three parasite-treated groups. Furthermore, within the coccidia-treated group, it was clearly observed that only the VP group with a flexible linker linked to TAT (i.e., the target protein of the present invention, APP) had a superior effect, while VP alone was ineffective. This suggests that VP exerts its antiprotozoal effect by transducing into cells (cells and protozoa) through TAT.

[0069] Example 5: Verification of the Antiprotozoal Effect of Broad-Spectrum Antiprotozoal Peptides in Vivo

[0070] (1) Experimental animal breeding

[0071] The experimental animals were chickens that were susceptible to Eimeria, Plasmodium, and Trichomonas. SPF chicken embryos were hatched in a sterile environment and raised in an environment free of Eimeria, Plasmodium, and Trichomonas, with free access to food and water.

[0072] (2) Drug treatment

[0073] After the broad-spectrum antiprotozoal polypeptide is freeze-dried, the test dose is 100 grams per ton of feed; the anticoccidial control drug is dinitramide, and the test dose is 125 grams per ton of feed; the antimalarial control drug is chloroquine phosphate, and the test dose is 250 grams per ton of feed; the anti-trichomonas control drug is dimetridazole, and the test dose is 400 grams per ton of feed.

[0074] (3) Insect attack and treatment grouping

[0075] Eimeria, Plasmodium and Trichomonas were cultured until sporulation and stored for future use.

[0076] SPF chicks were raised to 14 days of age. Overweight and underweight chicks were removed and weighed, and their initial weights were recorded. The chicks were randomly divided into groups of 10 chicks per group. Grouping was determined according to Table 1.

[0077] Table 1 Insect attack and treatment groups

[0078] At 15 days of age, all groups requiring challenge received an oral dose of 10,000 eggs of the corresponding protozoan species. Daily observation and recording of water intake, food intake, feces, and mortality were performed for each group. Dead birds were promptly dissected and the cause of death analyzed. Seven days after challenge, survival rates were recorded for each group. Birds that died en route were dissected and the cause of death analyzed. Surviving birds were sacrificed by cervical dislocation and weighed. The cecum was removed, and the mucosa and contents were scraped to determine the number of oocysts in each bird.

[0079] (4) Evaluation of antiprotozoal effects

[0080] The Antiprotozoan Index (API) was set as follows: API = (survival rate + relative weight gain rate) - (lesion value + oocyst value); survival rate = (number of surviving chickens at the end of the test / number of test chickens) × 100%; relative weight gain rate = (weight gain of the test group / weight gain of the blank control group) × 100%; API ≥ 160 was judged as excellent, API = 130-159 was judged as good, API = 100-129 was hyposensitive, and API < 100 was judged as invalid.

[0081] The lesion reduction rate (RLS) was set as follows: RLS = (mean lesion score of the control group without medication - mean lesion score of the group with medication) / mean lesion score of the control group without medication × 100%. RLS ≥ 50% was considered fully sensitive.

[0082] The antiprotozoal index (API) and RLS results of each group are shown in Table 2.

[0083] Table 2 Antiprotozoal index (API) and lesion score reduction rate (RLS) of each group

[0084] The per-chicken attack dose in this trial was relatively low. In terms of the Antiprotozoal Index (API), the broad-spectrum antiprotozoal peptide API values ​​of 169.1, 165.5, 159.9, and 149.8 for different protozoa were all considered good, exceeding the good level. These values ​​were 21%, 34%, 20%, and 60% higher than the three chemical agents, respectively. Broad-spectrum antiprotozoal peptides were highly sensitive to chemical agents in terms of RLS. Broad-spectrum antiprotozoal peptides generally showed better efficacy than chemical agents in terms of oocyst values. Broad-spectrum antiprotozoal peptides also showed better weight gain than chemical agents. Overall, broad-spectrum antiprotozoal peptides were superior to chemical antibiotics in terms of in vivo antiprotozoal efficacy.

[0085] Accordingly, the broad-spectrum antiprotozoal polypeptides of the present invention can be used to antiprotozoa in animals in vivo and / or in vitro. When an animal orally administers the broad-spectrum antiprotozoal polypeptides, the broad-spectrum antiprotozoal polypeptides can exert broad-spectrum antiprotozoal effects in the animal. When the broad-spectrum antiprotozoal polypeptides are administered to an animal in a breeding environment, the broad-spectrum antiprotozoal polypeptides can exert broad-spectrum antiprotozoal effects in the animal in vitro.

[0086] 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. A broad-spectrum antiprotozoal polypeptide, characterized in that The amino acid sequence of the broad-spectrum antiprotozoal polypeptide is composed of a transduction protein domain sequence, a linker protein sequence and a mutant APLV4-VP protein sequence connected in sequence; wherein the transduction protein domain sequence is an HIV-TAT transduction protein domain amino acid sequence, as shown in SEQ ID NO.1; and the linker protein sequence is a flexible linker protein amino acid sequence (GGGGS) n , n≥1, as shown in SEQ ID NO.2; the mutant APLV4-VP protein sequence includes a sequentially connected mutant VP4 protein amino acid sequence and original VP2, VP3, and VP1 protein amino acid sequences, and the complete amino acid sequence is shown in SEQ ID NO.

7.

2. A method for preparing a broad-spectrum antiprotozoal polypeptide as claimed in claim 1, characterized in that: The steps include: (1) Obtaining the gene sequence of broad-spectrum antiprotozoal polypeptides; (2) Using Bacillus subtilis as the expression host strain to construct an engineered bacterium; (3) Fermentation and purification to obtain broad-spectrum antiprotozoal polypeptides; the specific fermentation and purification method is as follows: ① Take the qualified engineered bacteria after sequencing and culture them in a shaker at 37℃ and 220rpm until OD600 is 2.0; add 3mM IPTG, then adjust the temperature of the shaker to 18℃, and harvest after 12h of induction; centrifuge at 4℃ and 12000rpm for 15min, take the supernatant and discard the precipitate; ② Take the supernatant after centrifugation and add 80% saturation (NH4)2SO4 in a 1:1 ratio; then place the mixture in a 4°C refrigerator for 48 hours to flocculate and precipitate the protein in the fermentation broth; centrifuge at 4°C and 12000rpm for 15 minutes, take the precipitate, and discard the supernatant; suspend the precipitate with PBS buffer, and dialyze with PBS of the same concentration as the suspension to remove salt; take the dialyzed protein solution and add 80% saturation (NH4)2SO4 in a 1:1 ratio; then place the mixture in a 4°C refrigerator for 48 hours to flocculate and precipitate the protein in the mixture; centrifuge at 4°C and 12000rpm for 15 minutes, take the precipitate, and discard the supernatant; suspend the precipitate with PBS buffer, and dialyze with PBS of the same concentration as the suspension to remove salt to obtain the target protein.

3. The method for preparing a broad-spectrum antiprotozoan polypeptide according to claim 2, characterized in that: The broad-spectrum antiprotozoal polypeptide gene sequence is obtained in step (1), and the specific method for obtaining the gene sequence is as follows: ① Obtain the transduction protein domain sequence; ② Obtain the mutant APLV4-VP protein sequence; ③ Using a linker protein sequence to connect the transduction protein domain sequence and the mutant APLV4-VP protein sequence to obtain an amino acid sequence of a broad-spectrum antiprotozoal polypeptide; ④ Use DNAMAN software for reverse translation, optimize it based on the codon preference of Bacillus subtilis, add restriction site BamH Ⅰ upstream and Xba Ⅰ downstream to obtain the gene sequence of the broad-spectrum antiprotozoal polypeptide.

4. The method for preparing a broad-spectrum antiprotozoal polypeptide according to claim 2, characterized in that: The engineering bacteria in step (2) are constructed by: double-digesting the obtained gene sequence of the broad-spectrum antiprotozoan polypeptide and the pHT43 plasmid with an endonuclease, connecting the double-digestion products with T4 ligase, picking positive single colonies, expanding and culturing at a constant temperature, and sending them for detection to obtain qualified ones.

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