CN112028995B - Polypeptide for resisting coccidiosis infection and application thereof - Google Patents

Abstract

The invention discloses a polypeptide for resisting coccidian infection and application thereof. According to the invention, the structure and antigenicity of seven repetitive domains of EtMIC3 are analyzed, then B, C1 two structural domains are selected for research, EtMIC3-BC1 protein is used as a target molecule, 7 affinity peptides capable of being specifically combined with the EtMIC3-BC1 protein are selected by a phage display technology, then three A peptides, D peptides and W peptides with good binding property are selected by ELISA and other methods, and the amino acid sequences of the A peptides, the D peptides and the W peptides are respectively shown in SEQ ID NO. 1-3. In vitro tests show that 3 polypeptides can effectively inhibit E.tenella sporozoites from invading MDBK cells in vitro; in vivo tests show that the chicken orally take 3 polypeptides corresponding to the bacteriophage to show the protective effect against coccidian infection. The invention further provides application of the polypeptide in preparing a medicine for preventing or treating coccidiosis infection.

Description

Polypeptide for resisting coccidiosis infection and application thereof

Technical Field

The invention relates to a polypeptide for resisting coccidium infection of chicken and application thereof, belonging to the field of polypeptide for resisting coccidium infection and application thereof.

Background

In the daily breeding process, the coccidiosis disease can be caused by the chicken ingesting water or food polluted by coccidian sporulated oocysts, all the day-old of each variety of chicken can be infected, and the chicken is susceptible to infection at the age of 3-6 weeks generally (Liu Jian Tao. comprehensive control technology of chicken coccidiosis [ J ]. contemporary livestock and poultry breeding, 2017, (10): 35.). After being ingested, the pathogenic coccidian oocysts are oozed under the action of a digestive system, and then the microspores secrete microline proteins, and when the sporozoites randomly collide with host cells, the microspores are combined with corresponding receptor molecules on the surfaces of the cells; when sporozoites adhere to the surface of a host cell, organelles such as rods and compact granules secrete invasion-related proteins and enzymes, thereby facilitating entry of the sporozoites into the host cell. Sporozoites entering host cells firstly travel to generation of schizogenesis, which seriously damages small intestine villus epithelial cells and causes pathological changes such as intestinal bleeding and inflammation. After the merogenesis, the sexual reproduction stage is carried out, and the large and small gametes developed by sporozoites are fused to form zygotes and further developed into mature oocysts which are discharged out of the body along with feces.

At present, chicken coccidiosis has great harm to poultry industry worldwide, causing huge economic loss, wherein the harm brought by E.tenella is the greatest. Currently, the feeding of anticoccidial drugs and the preparation of vaccines using live coccidia are the main methods for combating coccidiosis. But the sensitivity of the coccidia to the medicine is lower and lower in the process of using the anticoccidial medicine, the dosage of the medicine has to be increased, so that the coccidia generates the drug resistance and the treatment cost is increased; in the process of using the attenuated live vaccine to prevent coccidiosis, some chickens with poor body resistance or susceptibility to coccidiosis only have the phenomenon of coccidiosis infection, thereby losing the significance of preventing coccidiosis. At present, many researches are carried out from the aspects of important parasite antigen epitopes, immune recognition of specific protein molecules, interaction between antigens and antibodies, and the like, but no commercial novel vaccine is available at present. In recent years, researchers have become aware that the development of new coccidia vaccines requires the first clarification of the mechanisms of invasion by the individual stages of the worm. Research shows that the E.tenella microwire protein 3(E.tenella MIC3) plays an important role in the coccidium invasion process and can be used as a vaccine candidate protein for effectively preventing coccidium diseases. In the process of free collision between sporozoites and chicken cecal epithelial cells, EtMIC3 secreted on the surface of sporozoites by a microwire and receptor molecules on the surface of host cells are specifically combined, so that a precondition is provided for invasion of polypide into the host cells. EtMIC3 includes 7 domains (A, B, C1, C2, C3, C4, D), including the middle 4 completely conserved repeat domains (C1, C2, C3, C4) and two ends 3 incompletely conserved regions (A, B, D), and it is not clear which fragments play a major role in the invasion process of the insect body. The invasion process of E.tenella is very complicated, the invasion characteristic brings great difficulty for the treatment and prevention of coccidian diseases caused by the E.tenella, the discovery of proteins relatively conserved in the invasion and development stages of E.tenella is the key for preventing coccidian diseases, and the conserved proteins are developed into recombinant vaccine immune chicks through certain technical means, so that specific immune response of corresponding proteins can be generated in an organism to resist coccidian infection and protect the organism.

Phage display technologies (Phage display technologies) are a new biotechnology that can insert DNA sequences of other foreign proteins into the structural genes of Phage capsid proteins, and the inserted foreign genes are expressed along with the Phage capsid proteins, and the foreign proteins are displayed on the surface of Phage when the Phage is reassembled (Smothers JF, et al. Phage display: affinity selection from biological libraries [ J ]. Science,2002,298(5593):621 and 622). This technique was created by Smith et al in 1985 by inserting a foreign gene into the gene III of phage f1, and displaying the polypeptide encoded by this foreign gene on the surface of the phage as a fusion protein (Smith G P. structural fusion protein expression vectors connected with the virus surface [ J ] Science,1985,228(4705): 1315-1317.). By this technique, not only the specific protein required by people can be displayed on the surface of phage, but also the structural gene corresponding to the specific protein can be included in the core DNA of phage, so that the phage virion can reach the state of uniform genotype and phenotype, and the phage can display the foreign peptide or protein with independent spatial structure and biological activity (Pascke M. phase display systems and the third applications [ J ]. Applied Microbiology and Biotechnology,2006,70(1): 2-11.). Phage display technology also combines gene expression products with affinity screening (Smothers J F, Henikoff S, Carter P.Tech.Sight.Phage display.affinity selection from biological proteins [ J ]. Science,2002,298(5593):621-2.) by selecting appropriate proteins of interest and screening the phage display library according to phage screening procedures to obtain ligands that specifically bind to the proteins of interest. Then, the gene sequence and protein structure of the screened ligand are analyzed by related technical means, so that the action mechanism of the target protein and the ligand thereof can be more specifically understood. With the continuous improvement of phage display technology, the technology has penetrated into the research fields of intercellular signal transduction, gene expression and regulation, antibody preparation, drug screening, vaccine development, disease diagnosis and treatment, etc., and becomes an important technical means in many research fields (wangxin 29760, in pofeng, jiazhen, etc.. the overview and application of phage display technology [ J ]. the journal of chinese pharmacology and toxicology, 2019, 33 (10): 930..

The phage display technology has many advantages, such as the ability to analyze the molecular structure of antigen and antibody, identify epitope of antigen, screen out the affinity peptide of antigen or antibody, stable affinity, easy mass amplification, etc., so it has important role in the research and prevention of parasitic diseases. By screening antibody libraries with related proteins by phage display technology, the finally screened high affinity antibody mimetics can be applied to the diagnosis and treatment of some parasitic diseases. The phage display library or the antibody library is subjected to biological panning by using related antibodies or antibody preparations, related specifically-bound polypeptides can be screened, and the analysis of the structure and the function of the antigens related to the parasitic diseases is facilitated by performing sequence analysis, immunocompetence judgment and other treatments on the specifically-bound polypeptides.

Disclosure of Invention

The invention mainly aims to provide a polypeptide for resisting coccidian infection and a coding gene thereof;

the other purpose of the invention is to prepare the polypeptide or the coding gene thereof into a vaccine for preventing or treating chicken coccidiosis.

The above object of the present invention is achieved by the following technical solutions:

the invention takes EtMIC3-BC1 protein as a target molecule, two B, C1 fragments are selected for research through the structure of seven repetitive domains of EtMIC3, antigenicity analysis and the like, 7 affinity peptides capable of being specifically combined with the EtMIC3-BC1 protein are screened out through a phage display technology, then three A peptides, D peptides and W peptides (the amino acid sequences of which are respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3) which have higher affinity and can be specifically combined with the EtMIC3-BC1 protein are selected out through ELISA and other methods for artificial synthesis, and the degree of the inhibition of coccidian infection is further evaluated through in vitro experiments and in vivo experiments; in vitro tests show that the A peptide, the D peptide and the W peptide can inhibit E.tenella sporozoites from invading MDBK cells in vitro, wherein the A peptide and the D peptide have good effects; in vivo inhibition tests show that the A peptide, the D peptide and the W peptide have a certain protective effect on coccidium infection after being orally taken corresponding to the phage, and the phage corresponding to the A peptide and the D peptide have a good effect.

Therefore, the A peptide, the D peptide and the W peptide with the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively obtained by screening can be applied to coccidium infection resistance.

The invention firstly provides a polypeptide for resisting coccidian infection, and the amino acid sequence of the polypeptide is shown in (a) or (b): (a) selected from the group consisting of the polypeptides shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; or (b) protein variants which are derived from any one of the amino acids shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 by substitution, deletion or/and insertion of one or more amino acid residues and still have the function or activity of resisting coccidian infection.

The invention further provides a coding gene of the polypeptide and an expression vector containing the coding gene.

The A peptide, the D peptide or the W peptide is prepared into a vaccine according to a conventional vaccine preparation method, and the chicken is immunized by an oral administration or injection mode to only improve the coccidium infection resistance of the chicken.

For example, one skilled in the art can prepare peptide vaccines from peptides a, D or W (e.g., phage peptide vaccines using phage as a carrier, nanoparticle vaccines using nanoparticle packaging, oral lactic acid bacteria vaccines using lactic acid bacteria as a delivery carrier, etc.); or prepared as a nucleic acid vaccine according to conventional methods in the art, which may be a DNA vaccine comprising a plasmid having a promoter and appropriate transcriptional and translational control elements, and a nucleic acid sequence encoding the polypeptide. In some embodiments, the plasmid further comprises an enhancing element (e.g., a sequence for expression level, intracellular targeting, or proteasome processing). In some embodiments, the DNA vaccine comprises a viral vector comprising a nucleic acid sequence encoding a polypeptide. In some embodiments, the DNA vaccine is introduced into the animal through a needle, gene gun, aerosol syringe, patch, microneedle, abrasion, and other means. In some forms, the DNA vaccine is incorporated into liposomes or other forms of nanobodies. In some embodiments, the DNA vaccine comprises a delivery system selected from the group consisting of transfection agents, protamine liposomes, polysaccharide particles, cationic nanoemulsions, cationic polymers, cationic polymer liposomes, cationic nanoparticles, cationic lipid and cholesterol nanoparticles, cationic lipids, cholesterol and PEG nanoparticles, dendrimer nanoparticles. In some embodiments, the DNA vaccine is administered by inhalation or ingestion.

Administration is typically a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered treatment), which is sufficient to prevent or delay the onset of the disease or disorder. The dosage can be determined according to various parameters, in particular according to the substance used, the age, weight and condition of the animal, the route of administration and the desired protocol. The amount of antigen in each dose was selected as the amount that induced the immune response.

Routes of administration include, but are not limited to, oral, subcutaneous, intradermal, and intramuscular.

Detailed description of the invention

According to the invention, after the pET-30a-EtMIC3-BC1 recombinant protein expressed by pronucleus is purified and renatured, immunization is carried out on the New Zealand white rabbits in a mode of back subcutaneous multipoint injection. The indirect ELISA method detection result shows that the titer of the prepared polyclonal antiserum is 2¹⁶It is shown that the immunization program chosen and the amount of protein used for immunization are reasonable in the preparation of the polyclonal antibody. A Western blot method is adopted to detect the biological activity of the prepared EtMIC3-BC1 polyclonal antiserum. The result shows that the recombinant target protein and the sporozoite protein can be specifically combined with the polyclonal antiserum, and obvious bands appear at the position of 41kDa, which indicates that an antibody induced by the EtMIC3-BC1 recombinant protein induced by a prokaryotic expression system has good biological activity.

The invention takes the purified and renatured EtMIC3-BC1 recombinant protein as a target molecule, and totally carries out 4 rounds of biological screening according to a phage dodecapeptide library and a phage screening manual, thereby screening phage clone which can be specifically combined with the EtMIC3-BC1 recombinant protein. In the screening process, the same number of phages are respectively put into each round of biological screening, and the number of eluted phages shows an increasing trend along with the increase of the times of biological screening, which shows that the more the screening times are, the more phages capable of specifically binding with the target protein are. After the fourth screening, thirty well-conditioned plaques are randomly picked from an IPTG/Xgal plate for phage amplification, the binding capacity of 30 phage amplification supernatants and EtMIC3-BC1 protein is detected by an indirect ELISA method, 24 phage monoclonals with higher affinity are selected according to the ELISA result, and DNA of positive binding clones is extracted according to the method steps provided by a phage screening manual. The phage specificity primer sequences provided in the manual are used as an upstream template and a downstream template, each phage DNA is identified by PCR, the 1% gel electrophoresis result shows that the selected 24 phage single clones can amplify target fragments with the size of about 250bp, PCR products are recovered and sequenced, and the nucleotide sequences of the phage clones are deduced according to a simplified genetic code table, so that the amino acid sequences of each affinity phage are obtained. The analysis of the obtained amino acid sequences shows that the total number of the selected 24 affinity dodecapeptides is eight, and three sequences which have higher affinity with EtMIC3-BC1 and relatively more polypeptide sequences are selected through analysis and are respectively as follows: AGRLLTPTMSLV (A peptide), DYHDPSLPTLGK (D peptide), WKDVHKAWLLEP (W peptide). In order to further detect the three selected polypeptides, the binding capacity of the phage corresponding to the three polypeptides and EtMIC3-BC1 is detected by an ELISA method, and the result shows that the phage corresponding to the three polypeptides can be well bound with EtMIC3-BC 1; the rabbit anti-EtMIC 3-BC1 polyclonal antibody can be combined with three affinity phages competitively and combined with the EtMIC3-BC1 protein by performing a competition ELISA experiment by using the anti-EtMIC 3-BC1 polyclonal antibody, thereby indicating that a combination site similar to the anti-EtMIC 3-BC1 polyclonal antibody possibly exists on the sequence of the affinity phages; the sporozoite ultrasonic products are respectively subjected to ELISA reaction with three affinity phages, and the test result can analyze that the affinity phages can be combined with a certain concentration of EtMIC3 protein. The results of the affinity detection tests show that the three screened polypeptides have certain specificity, so that the polypeptides synthesized by the three sequences and having the purity of 95 percent are further subjected to in vitro coccidium resistance tests and in vivo coccidium resistance tests.

The invention proves that the three screened affinity polypeptides can be specifically combined with EtMIC3-BC1, so that the invention further discusses whether the three screened polypeptides can inhibit E.tenella sporozoites from invading MDBK cells as well as the anti-EtMIC 3-BC1 multi-resistance. In the in vitro experiment process, the sporozoites are fluorescently labeled with a CFDA-SE fluorescent probe capable of generating irreversible binding in the sporozoites before invading the MDBK cells, 300 mu g/mL rabbit anti-EtMIC 3-BC1 polyclonal antibody is used as a control, and the detection result is carried out by a flow cytometer after the invasion is finished. Experimental results show that the addition of the anti-EtMIC 3-BC1 polyclonal antibody can obviously inhibit the invasion of sporozoites to MDBK cells, and the inhibition rate reaches 75.2%, when the concentrations of the A peptide, the D peptide and the W peptide are respectively 25 mu g/mL, 50 mu g/mL, 75 mu g/mL, 100 mu g/mL and 125 mu g/mL (within the maximum nontoxic concentration range of the cells), the three polypeptides can inhibit the invasion of sporozoites to the MDBK cells to a certain extent, and when the concentration is 125 mu g/mL, the inhibition rates of the A peptide and the D peptide respectively reach 71.8% and 54.6%. The above test results all show that the three selected affinity peptides can inhibit the invasion of E.tenella sporozoites to MDBK cells in vitro to a certain extent.

The screened polypeptide is proved to be capable of inhibiting E.tenella sporozoites from invading MDBK cells to a certain extent in vitro, but whether the corresponding phages can also play a role in resisting coccidiosis in vivo is not clear, so the invention further judges the coccidiosis resisting role of the selected phages through oral administration of the corresponding phages to chickens infected with Eimeria tenella oocysts and through observation of the oocyst reduction rate, weight gain effect, caecum lesion score, gram oocyst number, ACI and caecum histopathology of the chickens.

In the development process of the coccidiosis in chickens, mature oocysts are swallowed and then enter the digestive tract, sporozoites escape from the oocysts through mechanical grinding of gastrointestinal tract peristalsis and chemical digestion of trypsin and bile, and enter intestinal epithelium for schizogenesis. From oocysts to ovaIt takes 2.5-3 days for the spore to crack to produce the first generation schizonts and develop into merozoites (suo xu, edited by Lizhou Qing dynasty, coccidiosis in chickens, China agriculture university Press, 199808.). In addition, among a plurality of vaccination modes of the vaccine, the oral vaccine has great temptation in vaccine research due to the characteristics of simple and convenient operation, low cost, convenient popularization and the like. Studies have shown that the bacteriophage can activate the innate and adaptive immune system to enhance the immunity of the organism (Krystina L, Hess C M, Jewell. Phage display as a tool for vaccine and immunotherapy)].Bioengineering& a study of the ability of phages to stimulate the body to produce an immune response after immunization as an oral vaccine has also been reported (Scott J K, Smith G P. searching for peptide ligands with an epitope library [ J.]Science,1990,249 (4967)). Therefore, the experiment is orally taken by 1X 10 chickens at 21 days of age⁴Each E.tenella sporulated oocyst is orally taken by 1 multiplied by 10 for 0-2 days after the attack of the insect¹²pfu corresponding phage was evaluated for the inhibition of invasion by the selected polypeptides, and two control groups of PBS and e.tenella were set. All chickens were killed by dissecting on the eighth day after the attack of the insects, and compared with the control group of the attack of the insects, the weight gains of the A group, the D group and the W group were more, the caecum lesion symptoms were relatively lighter, the number of oocysts discharged was less, the ACIs of the A group and the D group were 168.74 and 161.27 respectively, the anticoccidial effect was better, the ACI of the W group was 153.64, and the anticoccidial effect was relatively weaker. The test result proves that the three phages selected by oral administration have a certain degree of coccidian infection resistance in vivo, and the vaccine form also has important significance in the practical application of diseases.

The terms and definitions to which this invention relates

The term "polypeptide" refers to a full-length protein, a portion of a protein, or a peptide characterized by a string of amino acids.

The term "fragment" or "fragment of a polypeptide" refers to a string of amino acids or a sequence of amino acids that are generally of reduced length relative to the above-mentioned or one reference polypeptide and that comprise, on a common portion, the same sequence of amino acids as the reference polypeptide.

The term "active ingredient" refers to a polypeptide intended to induce an immune response, and may include the polypeptide product of a vaccine or immunotherapy composition produced in an animal to which it is administered.

The polypeptide compositions disclosed herein may comprise one or more "pharmaceutically acceptable carriers". These carriers are typically large, slowly metabolized macromolecules such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al, 2001, Vaccine,19:2118), trehalose (WO 00/56365), lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such vectors are well known to those of ordinary skill in the art. The pharmaceutical composition may also contain diluents such as water, saline, glycerin, and the like. In addition, auxiliary substances may be present, such as wetting or emulsifying agents, pH buffering substances and the like. Sterile pyrogen-free phosphate buffered saline is a typical vehicle.

Suitable adjuvants include aluminium salts such AS aluminium hydroxide or aluminium phosphate, but may also be salts of calcium, iron or zinc, or may be insoluble suspensions of acylated tyrosine or acylated sugars, or may be cationically or anionically derivatised sugars, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g. reduced toxicity), 3-O-deacylated monophosphoryl lipid A (3D-MPL), QuilA, saponins, QS21, Freund's incomplete adjuvant, Merck adjuvant 65, AS-2, CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides or imidazoquinolone compounds (e.g. imiquimod and its homologues).

Drawings

FIG. 1 recombinant plasmid pUC57-EtMIC3-BC1 PCR identification; m: DNA molecular mass standard; 1: PCR products; 2: and (4) water control.

FIG. 2 PCR identification of pET30a-EtMIC3-BC1 recombinant plasmid; m is DNA molecular mass standard; 1: PCR products; 2: and (4) water control.

FIG. 3 SDS-PAGE analysis of EtMIC3-BC1 protein expression; m: high molecular mass protein standard; 1: pET-30a empty vector; 2-5: inducing the recombinant bacteria for 0-3 h; 6: purified EtMIC3-BC1 protein.

FIG. 4Western blot to detect the specificity of polyclonal antibody against EtMIC3-BC1 protein; m: high molecular protein quality standard; 1: EtMIC3-BC1 recombinant protein; 2: sporozoite protein.

FIG. 5 monoclonal plaques on IPTG/Xgal/LB plates.

FIG. 6 ELISA detection of phage binding clones with EtMIC3-BC1 recombinant protein; BC: blank control; NC: negative control; 1-30: numbering the plaques; PC: and (4) positive control.

FIG. 7 phage PCR amplification products; m: DL 2000 DNA Marker; 1-24: corresponding to 1-24 phage PCR purified products respectively.

FIG. 8 results of ELISA analysis of phage binding to EtMIC3-BC1 recombinant protein.

FIG. 9 inhibition of phage binding to EtMIC3-BC1 recombinant protein by EtMIC3-BC1 polyclonal antibody.

FIG. 10 ELISA detects the binding of sporozoite EtMIC3 protein to phage.

FIG. 11 E. purification and fluorescent labeling of Tenella sporozoites; a: purified sporozoites; b: fluorescently labeled sporozoites.

FIG. 12 MTT assay measures the maximum non-toxic concentration of three polypeptides (A, D, W) on MDBK cells.

Figure 13 differential dilution of EtMIC3-BC1 polyclonal antibody inhibits e.tenella sporozoites from invading MDBK cells.

Figure 14 inhibitory effect of different concentrations of polypeptide on e.tenella sporozoites invasion of MDBK cells.

Figure 15 group chicken ceca lesion scores.

FIG. 16 reduction rate of oocyst discharge in each group of chickens.

FIG. 17 sets of cecum ocular pathophysiological changes; 1-2: a PBS group; 3-4: group A; a5-6: group D; a7-8: group W; a9-10: attack worm control group.

FIG. 18 is a view of the pathological tissues of the caecum of each group; a: pathological changes of the cecum tissue in the PBS group; b: pathological changes of cecal tissues in group A; c: group D pathological changes of cecum tissue; d: pathological changes of cecum tissue in W groups; e: and E, tentella attack worm control group cecal tissue pathological changes.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the embodiments are illustrative only and are not to be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test example 1 screening and identification of EtMIC3-BC1 affinity peptides

1. Test method

1.1 construction of pET-30a-EtMIC3-BC1 recombinant plasmid

1.1.1 Synthesis of the Gene of interest

The full length of the repeat domain gene of the EtMIC3-BC1 fragment was 846bp, synthesized by Shanghai Biotech, Inc., cloned into pUC57, and the restriction sites BamH I and Xho I were introduced. The delivery sample was E.coli Top10 Glycerol (containing the plasmid pUC57-EtMIC3-BC 1).

1.1.2 pUC-57-EtMIC3-BC1 plasmid extraction

The gene synthesis company delivered Top10 glycerol bacteria activated on Amp resistant LB plate, in the clean bench to pick single colony inoculated in LB liquid medium (A)⁺) In the medium, the plasmid was extracted from the overnight-cultured broth by shaking at 180rpm in a shaker at 37 ℃.

1.1.3 PCR identification and purification recovery of EtMIC3-BC1 Gene

PCR identification of the target gene: respectively taking front and back 18bp of a target gene fragment as upstream and downstream primers to carry out primer synthesis. F: ATGACCCTGCAGGAAGCG, respectively; r: GCTACCGCTCGGATCTTC are provided. The PCR result was identified by 1% agarose gel electrophoresis (m/v), and after the position of the band size was confirmed to be correct, the plasmid pUC57-EtMIC3-BC1 was double digested by two restriction enzymes BamH I and XhoI, followed by purification and recovery of the desired gene in large quantities.

Purification and recovery of target genes: the digested product was mixed with 1/10 volumes of 10 Xloading Buffer, and subjected to electrophoresis by adding to 0.8% agarose gel (m/v). The gel strips containing the target gene were cut rapidly under UV irradiation, collected in a weighed 2mL EP tube, and the target gene was recovered using a DNA gel recovery kit according to the instructions.

1.1.4 ligation of target Gene to expression vector

Taking E.coli/pET-30a bacterial liquid frozen at-80 ℃ to Kana⁺Resistant LB plate streaking, inverting the plate in a 37 ℃ constant temperature incubator for 12h, picking single colony in a clean bench and inoculating to 4mL LB liquid medium (K)⁺) In the culture, shaking and culturing the strain for 12 hours at 37 ℃ and 180rpm by a shaking table, and extracting the plasmid from the strain liquid according to the 1.1.2 plasmid extraction step. PCR identification of the extracted pET-30a plasmid was performed, the position of the band size was determined to be correct, double digestion of pUC57-EtMIC3-BC1 plasmid was performed with two restriction enzymes BamHI and XhoI, vector recovery was performed using a DNA purification recovery kit, and the recovered EtMIC3-BC1 fragment was ligated to the vector (16 ℃ overnight).

1.1.5 transformation of recombinant plasmids

1.1.6 extraction and identification of recombinant plasmids

Extracting recombinant plasmids: on plates coated with the transformant virus and incubated overnight, single colonies of good quality were selected and inoculated into Kana⁺And (3) performing shake culture overnight at 37 ℃ in a resistant LB liquid culture medium, and extracting recombinant plasmids in the bacterial liquid according to a plasmid extraction step. PCR identification of recombinant plasmids: the recombinant plasmid extracted above was preliminarily identified by PCR. And (3) PCR reaction conditions: the annealing temperature was 60 ℃ and the extension was 50S, and the total of 30 cycles was performed, and after the amplification was completed, 4. mu.L of the PCR product was taken out and the results were observed by 1% agarose gel electrophoresis. Double restriction enzyme identification of recombinant plasmid: the recombinant plasmid was double digested with BamH I and Xho I, respectively.

1.2 inducible expression of the recombinant plasmid pET-30a-EtMIC3-BC1 in an E.coli expression System

The positive recombinant plasmid pET30a-EtMIC3-BC1 was transformed into competent cells expressing E.coli BL21 and cultured overnight at 37 ℃. And (3) selecting a single high-quality positive plasmid transformation colony, inoculating the colony in 30mL LB liquid medium containing Kana +, and carrying out shaking culture in a shaker at 37 ℃ for 5-7 h at 180 rpm. Then, the OD value of the bacterial liquid is detected at the wavelength of 600nm, when the OD value of the bacterial liquid reaches 0.5, 1mL of recombinant bacterial liquid culture is sucked out and used as a control group before induction in an EP tube, IPTG is added into the rest culture until the final concentration is 1mmol/L, and the shaking culture is continued in a shaking table at the temperature of 37 ℃. And 1mL of the sample was taken out every 1h, placed in a refrigerator at 4 ℃ for temporary storage, and left for electrophoretic analysis. The microbial protein containing pET30a empty vector was analyzed by SDS-PAGE as a blank.

1.3 purification recovery and renaturation of EtMIC3-BC1 recombinant protein

1.3.1 purification of recombinant proteins

The collected sonicated pellet was suspended in PBS and mixed with 2 XSDS loading Buffer at a ratio of 1:1 and boiled in boiling water for 10 min. After cooling, the EtMIC3-BC1 recombinant protein samples were applied to a previously prepared 1.5mm thick gel plate for SDS-PAGE (5% gel concentrate, 8% gel isolate) electrophoresis. After bromophenol blue enters the electrophoresis solution, the electrophoresis apparatus is closed, the gel plate is taken out, the gel is carefully taken out by the gel plate under the flushing of fine water flow and is placed in PBS for cleaning, then the gel is moved into 0.25mol/L KC1 solution precooled at 4 ℃ for color development, when milky target bands appear on the gel, the target bands are quickly cut off, and redundant gel blocks are cut off as much as possible. Carefully cleaning a gel strip containing the target protein by PBS, grinding, adding a proper amount of PBS when the adhesive tape is ground into fine particles, uniformly mixing, freezing in a refrigerator at-40 ℃, dissolving, repeatedly freezing and thawing for three times, placing an EP tube in a centrifuge at 4 ℃ for 10min at 5000rpm, and collecting supernatant and storing at-80 ℃.

1.3.2 renaturation of purified proteins

Renaturation of the purified recombinant protein was carried out by dialysis and finally the renatured protein was taken out and analysed by SDS-PAGE.

1.4 preparation of polyclonal antiserum and identification thereof

1.4.1 preparation of polyclonal antisera

(1) Blood was collected through the rabbit ear vein and serum was isolated as a negative control; (2) firstly, exempting from: 2mg of renatured EtMIC3-BC1 recombinant protein is mixed with Freund's complete adjuvant according to the proportion of 1:1 and emulsified into oil emulsion, and the emulsified protein adjuvant mixture is used for immunizing New Zealand white rabbits by a back subcutaneous multipoint injection method. (3) And (2) avoiding: two weeks after the initial immunization, 1mg of renatured recombinant protein was emulsified with an equal volume of incomplete Freund's adjuvant and the immunization was performed as above. (4) And (3) three-step (I): three immunizations were performed one week after two immunizations, and the immunization dose and method were identical to the two immunizations. (5) Fourthly, avoiding: after one week of three immunization, four immunizations were performed, and blood was collected again through the auricular vein to measure the serum titer. (6) Serum was isolated from new zealand white rabbit blood obtained by cardiac bleeds one week after four immunizations.

1.4.2 Indirect ELISA for the detection of the titer of polyclonal antisera

By protein dilution (0.1mol/L pH 8.6 NaHCO)₃) EtMIC3-BC1 recombinant protein was diluted to 100. mu.g/mL concentration and used as antigen-coated ELISA plates at 100. mu.L per well. Serum and serum to be tested separated before immunization according to 2^-6、2^-7、2^-8…2^-17The dilution of (3) was diluted as a primary antibody and preimmune serum was used as a negative control.

1.4.3 Western-blot detection of the specificity of polyclonal antibodies

Western-blot detection was performed using sporozoite proteins and prepared EtMIC3-BC1 polyclonal antiserum.

1.5 biopanning of phage random dodecapeptide library against EtMIC3-BC1 protein affinity ligand

According to the phage surface display peptide library rapid screening peptide ligand use manual, four rounds of biological screening are carried out on a phage dodecapeptide library by taking EtMIC3-BC1 recombinant protein as a target molecule, all steps are strictly carried out according to aseptic operation, and the steps are simply described as follows:

1.5.1 first round of biopanning

(1) Protein was reconstituted in a clean bench with EtMIC3-BC1 with a 0.22 μm pore size and coated with coating solution (0.1mol/L NaHCO)₃pH 8.6) to a concentration of 100. mu.g/mL. The following steps are all performed by using a suction head with a filter element and performing aseptic operation; (2) coating the diluted protein on ELISA plate hole sterilized by ultraviolet irradiationMedium (100. mu.L/well), coated overnight at 4 ℃; (3) discard the coating solution, patting the plate hard on a sterile paper towel, and removing the residual liquid in the holes. Adding blocking liquid (200 μ L/hole), and sealing at 4 deg.C for 3 hr; (4) and (4) reversely beating and throwing the ELISA plate for many times, and discarding the blocking liquid as much as possible. To the well was added 200. mu.L of TBST (TBS + 0.1% [ v/v ]]Tween-20) is quickly washed for 6 times, the ELISA plate needs to be shaken for multiple times during washing, the residual blocking liquid in the holes is sufficiently washed away, the buffer solution is discarded, and the plate is inverted and thrown on a clean paper towel to remove the residual buffer solution; (5) 1.5X 10 of phage from the original library were removed by 100. mu.L of 0.1% TBST buffered dilutions¹¹pfu phage was diluted and added to the ELISA wells at one time, the ELISA plate was blocked, the plate was placed on an oscillator and shaken slowly at room temperature for 30 min; (6) pouring out the unbound phage, and turning the ELISA plate upside down on a clean sterile paper towel to forcibly flap and remove the unbound phage; (7) rapidly washing the plate 10 times with 0.1% TBST buffer solution, and replacing paper towels as much as possible during plate beating in order to avoid cross contamination; (8) absorbing 100 mu L of eluent (0.2mol/L Glycine-HCl pH 2.2) and adding the eluent into an ELISA hole, gently shaking the eluent for 35min at room temperature, collecting the eluent and placing the eluent in a sterile EP tube, and adding 15 mu L of 1mol/L Tris-HCl with pH of 9.1 for neutralization; this mixture was the first round of phage selection product and stored at 4 ℃.

1.5.2 phage titer assay

(1) Taking out the E.coli ER2738 stored in a refrigerator at-80 ℃ and marking on an LB-Tet plate by using an inoculating loop, and inversely placing the plate in the incubator at 37 ℃ for culturing for 12 hours; (2) inoculating a single colony in a good sterile picking state into a sterile test tube containing 3mL of LB-Tet liquid culture medium, and culturing for 4.5-5 h at 180rpm in a 37 ℃ incubator to enable the OD value to reach 0.5 at the wavelength of 600 nm; (3) phage selection from each round of selection was performed using Low LB broth 10^-1～10^-5Diluting by multiple times; (4) 1mL of the cultured thallus culture in the middle logarithmic phase is taken and distributed into five sterile EP tubes (200 mu L/tube), 10 mu L of phages with different dilutions are respectively added into the tubes, the mixture is inverted and mixed evenly, and the mixture is kept for 10min at room temperature. Add to centrifuge tubes containing equal amounts of bacterial culture. Oscillating and mixing, standing at room temperature for 2 min; (5) mixing the above five EP tubesThe mixture was transferred sequentially into tubes containing 4mL of top agar medium at about 45 ℃ one tube per dilution, mixed and quickly poured onto LB/IPTG/Xgal plates preheated to 37 ℃, the plates were shaken slowly to allow the top agar to spread evenly, and each dilution was poured onto one plate and labeled. When the plate is completely solidified, the plate is placed in an incubator at 37 ℃ upside down and is cultivated overnight in a dark place; (6) plates with the number of the phage blue spots not more than 100 are counted, and the titer is calculated according to a phage titer calculation formula. The formula: the titer of phage eluate was obtained from plaque forming unit (pfu) titer per 10. mu.L phage (number of plaques × dilution factor).

1.5.3 amplification of first round elutriation eluate

(1) Taking 200 mu L of E.coli ER2738 bacterial culture cultured overnight and 50 mu L of first-round elution product aseptically, inoculating the bacterial culture and the first-round elution product into 20mL of Low LB liquid culture medium, and culturing for 4.5-5 h in a shaker at 37 ℃ at 180 rpm; (2) and (3) aseptically subpackaging the bacterial liquid cultured in the step (1) into centrifuge tubes, and centrifuging for 15min at 8000rpm in a 4 ℃ centrifuge. Collecting the supernatant into another fresh sterile centrifuge tube, and centrifuging again; (3) the 80% centrifuged supernatant was placed in another clean sterile centrifuge tube and 1/6 volumes of PEG/NaCl was added to the supernatant. Placing the mixture into a refrigerator at 4 ℃ for settling overnight; (4) the phage after overnight sedimentation was centrifuged at 10000rpm in a 4 ℃ centrifuge for 15min, and the supernatant was discarded. Instantly separating to remove the residual supernatant; (5) suspending the precipitate in a centrifuge tube with 1mL TBS, mixing well, transferring into another new centrifuge tube of 2mL, centrifuging at 8000rpm at 4 deg.C for 10min to remove residual substances; (6) the supernatant was transferred to another fresh sterile 1.5mL EP tube and a second sedimentation on ice was performed for 1h with additional 1/6 supernatant volumes of PEG/NaCl; (7) centrifuging the phage subjected to secondary sedimentation in a 4 ℃ centrifuge at 10000rpm for 15min, discarding the supernatant, performing instantaneous separation, and removing the residual supernatant; (8) the pellet was suspended well in 200. mu.L TBS and centrifuged at 8000rpm for 1min at 4 ℃ in a centrifuge to remove remaining insoluble impurities. Transferring the supernatant into a new 1.5mL EP tube, wherein the product is the amplification product of the eluate; (9) 10 μ L of the amplification product was collected and weighed as 10^-8-10^-11Is diluted in two-fold ratio, and the titer of the amplified product of the phage eluate is determined according to the titer determination procedure 2.2.5.2。

1.5.4 second, third and fourth rounds of biological elutriation

The concentration of Tween-20 in TBST of 1.5.1 was adjusted to 0.5%, the number of plate washes was increased to 20, and the screening procedure was the same as that of 1.5.1.

1.5.5 amplification of second, third, and fourth rounds of eluate

Amplification step 1.5.3 same, the fourth round of bioscreening eluate amplification product was subjected to 10^-8～10^-12And the titer of the eluate amplification product of the fourth round is determined, and the plate with the number of the phage blue spots less than 100 is stored in a refrigerator at 4 ℃ in a dark place.

1.6 characterization of binding clones

1.6.1 amplification of plaques

(1) Aseptically picking a single E.coli ER2738 colony to inoculate into LB-Tet liquid culture medium, and culturing at 37 ℃ overnight at 180rpm in a shaking table; (2) inoculating 10 mu L of E.coli ER2738 after overnight culture into a test tube filled with 1mL of Low LB liquid culture medium, and cloning 1 tube for each phage to be identified; (3) picking up 30 plaques (each plaque is separated by a certain distance) on an LB/IPTG/Xgal plate with blue plaques not more than 100 which are stored in a refrigerator at 4 ℃ in an ultra-clean bench when the titer of the eluate amplification products of the fourth round of screening is determined, and sequentially inoculating the plaques into the test tube, wherein one tube of each plaque is one tube. Culturing for 4.5-5 h at 37 ℃ and 180 rpm; (4) the cultures were aseptically transferred to 2mL clean sterile centrifuge tubes and centrifuged at 8000rpm for 15min at 4 ℃ in a centrifuge. Transferring 80% of the supernatant after centrifugation to another new sterile centrifuge tube, and standing at 4 deg.C for short time or mixing 1:1 with sterilized glycerol and storing at-20 deg.C for long time.

1.6.2 ELISA detection of affinity phage binding to EtMIC3-BC1 protein

(1) EtMIC3-BC1 recombinant protein was treated with 0.1mol/L NaHCO at pH 8.6₃Diluted to 100. mu.g/mL, coated in ELISA plates (100. mu.L/well) and coated overnight in a 4 ℃ freezer; (2) discard the excess solution in the well, snap the plate upside down onto a newspaper and remove the residue. With PBST (0.5% [ w/v ]]Tween-20) the ELISA plates were washed with shaking 3 times (200. mu.L/well), 5min for each wash; (3) add 200 to each wellPutting mu L of 5% skim milk in an incubator at 37 ℃ for 2 h; (4) removing the sealing liquid, forcibly patting and throwing to remove residual liquid as much as possible, and washing the plate for 3 times by PBST; (5) adding 100 mu L of corresponding phage amplification supernatant into each hole of the ELISA plate, and putting the plate into a 37 ℃ incubator for incubation for 1 h; (6) discarding the excess solution, and washing the plate for 3 times with PBST; (7) diluting phage M13 polyclonal antibody (1:1000), adding 100 μ L diluted antibody into each well, incubating at 37 deg.C for 1 h; (8) pouring off the redundant solution, and washing the plate for 3 times by PBST; (9) adding 100 μ L of goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) diluted at a ratio of 1:5000 into each well, and incubating at 37 deg.C for 1 h; (10) the ELISA plate was developed with a fresh OPD developing solution in a dark box for 10min (100. mu.L/well) and measured for OD490nm with a microplate reader.

1.6.3 Rapid purification of affinity phage DNA

1.6.4 sequencing of phage panning

The phage specificity primer sequence provided by phage surface display peptide library rapid screening peptide ligand instruction manual synthesized by Suzhou Jinweizhi limited company is as follows: +130M13: 5'-TCACCTCGAAAGCAAGCTGA-3'; 28M13: 5'-CCCTCATAGTTAGCGTAACG-3'.

And (3) amplifying by PCR by using DNA purified after the amplification of the phage spot with higher affinity as a template. The PCR reaction conditions were pre-denaturation (95 ℃ 30s) → denaturation (94 ℃ 30s) → annealing (57 ℃ 30s) → extension (72 ℃ 30s) → final extension (72 ℃ 7 min). The PCR product was identified by electrophoresis on 1% agarose gel and the result of electrophoresis was observed. The DNA sequence of the PCR purified product was determined by +130M13 sequencing primer.

1.7 derivation and Synthesis of amino acid sequences of affinity phages

1.7.1 derivation of amino acid sequences of affinity phages

Fragments containing the nucleotide sequence of the selected peptide ligand amplified by phage-specific primers provided in the manual were carefully aligned with the 20 amino acids encoded by 32 codons and the nucleotide sequences were translated into the corresponding amino acid sequences by bioinformatics software according to the reduced genetic codon table.

1.7.2 Synthesis of amino acid sequences of affinity phages

The deduced amino acid sequence of dodecapeptide was used to synthesize polypeptide with a purity of 95% by bio-corporation.

1.8 ELISA was performed to detect the binding between affinity phage and recombinant EtMIC3-BC1 protein and the body EtMIC3 protein

1.8.1 ELISA detection of affinity phage binding to EtMIC3-BC1 recombinant protein

(1) Diluting phage corresponding to three polypeptides to 10 with diluent¹²pfu/100uL, coated in ELISA plates (100. mu.L/well), while the same titer of irrelevant phage was coated in the plates as control wells and overnight in a 4 ℃ freezer. Each sample is provided with two multiple holes; (2) the excess solution was poured off, the plate was patted upside down several times, the residue was removed thoroughly, and PBST (0.5% [ w/v ]]Tween-20) shake wash plate 3 times (200. mu.L/well), 10min each time; (3) blocking with 5% skim milk (PBS diluted) for 2h (200. mu.L/well) at 37 ℃; (4) discarding skim milk, and washing the plate with 0.5% PBST for 3 times; (5) 0.1mg/mL EtMIC3-BC1 recombinant protein (100 mu L/well) is added into the well and acted for 1h in an incubator at 37 ℃; (6) discard solution, wash plate 3 times with 0.5% PBST; (7) rabbit anti-EtMIC 3-BC1 polyclonal antibody (100 mu L/well) diluted at a ratio of 1:1000 was added to the wells and allowed to act for 1h at 37 ℃; (8) discard the well solution, wash the plate 3 times with 0.5% PBST; (9) HRP-labeled goat anti-rabbit IgG (100. mu.L/well) diluted at a ratio of 1:5000 was added to the wells, and incubated at 37 ℃ for 1 h; (10) the ELISA plate was developed with the OPD developing solution in place for 10min (100. mu.L/well) in a dark box, and OD490nm was measured with a microplate reader.

1.8.2 ELISA detection of inhibition of anti-EtMIC 3-BC1 multiple antibodies against affinity phage binding to EtMIC3-BC1

(1) Phage corresponding to the selected synthetic peptide were incubated with 0.1mol/L NaHCO at pH 8.6₃Diluting to 10¹²pfu/100uL, 100. mu.L per well coated ELISA plates (A plates) and coated overnight at 4 ℃. Each sample is provided with two compound holes; (2) the next day, another new ELISA plate (B plate) was blocked with 5% skim milk at 37 ℃ for 2 h; (3) the liquid in both plates was discarded A, B at the same time, and the residual liquid was removed by vigorous shaking. Both plates were washed 3 times (200. mu.L/well) with 0.5% PBST shaking for 10min each; (4) the A plate was blocked with 5% skim milk at 37 ℃ for 2h (200. mu.L/well). 50 mu L of EtMIC3-BC1 with the concentration of 0.1mg/mL is added into a plate hole of a B plate for recombinationProtein, rabbit anti-EtMIC 3-BC1 polyclonal antibody (the concentration is about 60, 40, 30, 24 and 20 mu g/mL) diluted in a ratio of 1: 10001: 15001: 20001: 25001: 3000 is added at the same time, 50 mu L of the polyclonal antibody is added in each hole, and the polyclonal antibody is incubated for 2h at 4 ℃; (5) the blocking solution was discarded from plate A and the plate was washed 3 times with 0.5% PBST; (6) adding 100 μ L of EtMIC3-BC1 recombinant protein premixed from the B plate and a polyclonal mixture of rabbit anti-recombinant protein to each well of the A plate, and incubating for 1h at 37 ℃; (7) discard the liquid in the wells, wash the plate 3 times with 0.5% PBST; (8) diluting rabbit anti-EtMIC 3-BC1 polyclonal antibody according to the proportion of 1:1000, adding 100 mu L of rabbit anti-EtMIC 3-BC1 polyclonal antibody into each hole, and incubating for 1h at 37 ℃; (9) discard the liquid in the wells, wash the plate 3 times with 0.5% PBST; (10) adding 100 μ LHRP-labeled goat anti-rabbit IgG (1:5000) to each well, and incubating at 37 deg.C for 1 h; (11) the ELISA plate was developed with a fresh OPD developing solution in a dark box for 10min (100. mu.L/well) and measured for OD490nm with a microplate reader.

1.8.3 ELISA for detecting the binding condition of affinity phage and insect body EtMIC3 protein

(1) All the phage were diluted to 1X 10 with diluent¹²pfu/100uL, coated in ELISA plate wells, 100. mu.L per well, coated overnight in a 4 ℃ freezer. Combining each phage and EtMIC3 recombinant protein into positive control and taking irrelevant phage as negative control, and setting two multiple holes for each sample; (2) discarding the solution in the wells, placing the ELISA plate upside down on the newspaper, shaking and throwing with force to remove the residual liquid sufficiently, and washing the plate with 0.5% PBST (200 μ L/well) on a shaker for 3 times, each time for 10 min; (3) add 200. mu.L of 5% skim milk (PBS diluted) to each ELISA well and block for 2h at 37 ℃; (4) discarding the blocking solution, placing the ELISA plate on an oscillator with 0.5% PBST, and washing the plate by oscillation for 3 times; (5) will be 1 × 10⁶A post-sonication solution of sporozoites (approximately 100. mu.L) was added to each well, and 100. mu.L of EtMIC3-BC1 recombinant protein at a concentration of 0.1mg/mL was added to the positive control wells and incubated for 1h at 37 ℃; (6) discarding the excess solution in the wells, washing the plate 3 times with 0.5% PBST; (7) adding 100 μ L rabbit anti-EtMIC 3-BC1 polyclonal antibody diluted according to the proportion of 1:1000 into each hole, and acting for 1h at 37 ℃; (8) discard the excess solution in the wells and wash the plate 3 times with 0.5% PBST; (9) adding 100 μ L diluted HRP-labeled goat anti-rabbit IgG (1:5000) to each well, and incubating at 37 deg.C for 1 h; (10) the ELISA plate was developed for 10min (10 min) with the in-situ OPD developer in a dark box0 μ L/well), OD490nm was measured with a plate reader.

2. Test results

2.1 construction of recombinant plasmid pET30a-EtMIC3-BC1

2.1.1 PCR identification of EtMIC3-BC1 Gene

After activating the positive escherichia coli containing the target gene fragment, carrying out plasmid extraction, and identifying through PCR, a brighter target band can be observed at a position with the size of 864bp in FIG. 1, and the result is consistent with the expected result. 2.1.2 PCR identification of recombinant plasmid pET30a-EtMIC3-BC1

After the prepared vector and the target gene are connected and transformed, single colony suspected to be positive is inoculated and cultured, then the bacterial liquid is subjected to plasmid extraction, and the plasmid is subjected to PCR identification, wherein the position of a target band is consistent with the expected size and is about 864bp (figure 2).

2.1.3 prokaryotic expression and purification of EtMIC3-BC1 recombinant protein

The correctly identified recombinant prokaryotic expression plasmid pET30a-EtMIC3-BC1 is transformed into E.coli BL21 competent cells, and 1.0mM IPTG is used for inducing for 3h under the condition of 180rpm in a shaker at 37 ℃ (shown in figure 3), and the result shows that a more obvious target strip can be observed at 41kDa, which accords with the prediction of protein size, and the expression quantity of the target protein reaches the maximum when the target protein is induced for 3 h. By performing a large amount of purification and recovery of recombinant proteins produced by recombinant cells after induced expression by cutting out SDS-PAGE gel strips containing the target proteins, and comparing the SDS-PAE patterns of the purified proteins with those of unpurified bacterial proteins, it was observed that the target proteins after purification were single in band and the purified products were the expected proteins.

2.2 potency and specificity determination of EtMIC3-BC1 polyclonal antisera

New Zealand white rabbits were immunized with the purified and renatured EtMIC3-BC1 protein to give rabbit anti-EtMIC 3-BC1 multi-antiserum. The detection result of the indirect ELISA method shows that the rabbit anti-EtMIC 3-BC1 multi-antibody titer is 2¹⁶This indicates that the antiserum had good reactivity. With EtMIC3-BC1 recombinant protein as a positive control, sporozoite ultrasonic protein was separated by SDS-PAGE and WBanti-EtMIC 3-BC1 polyclonal antibody was identified as the primary antibody and HRP-labeled goat anti-rabbit IgG was identified as the secondary antibody, with a specific band at the 41kDa position and consistent with the expected size of the protein of interest (FIG. 4).

2.3 biological screening of EtMIC3-BC1 recombinant protein affinity ligands

2.3.1 four rounds of screening and titer determination of the phage dodecapeptide library by EtMIC3-BC1 recombinant protein

Four rounds of biopanning of the phage random dodecapeptide library were performed using EtMIC3-BC1 recombinant protein as the target protein. And calculating the phage titer of the elution product and the elution amplification product of each round of biological screening according to the plaque number of the plate with the total plaque number less than 100 determined on the LB/IPTG/Xgal plate by the elution product of each round and the corresponding dilution factor (Table 1).

TABLE 1 phage elutriation product titer pfu assay

2.3.2 ELISA identification of affinity phages

When 30 fourth rounds of eluate amplification products were aseptically picked at random for titer determination, plaques on plates (FIG. 5) with no more than 100 blue plaques on LB/IPTG/Xgal plates were amplified (numbered 1-30), and the amplified supernatant was subjected to preliminary detection of specific binding of the amplified supernatant to the target protein by indirect ELISA using an anti-phage M13 antibody. The phage supernatant corresponding to the recombinant protein heteroconjugate polypeptide L peptide of EctoAMA1 is used as a positive control; the phage supernatants selected for the G protein of Vesicular Stomatitis Virus (VSV) served as negative controls. ELISA results showed that 24 of the 30 phage clones selected were able to bind specifically well to EtMIC3-BC1 recombinant protein (FIG. 6).

2.3.3 sequencing of affinity phages

The +130M13 and-28M 13 were used as upstream and downstream primers, and 24 plaque DNA purified products with higher affinity were used as templates for amplification, and the amplification results were observed by 1% agarose gel electrophoresis, which showed that the target fragments appeared clearly at about 250bp positions, corresponding to the expected positions (FIG. 7).

The nucleotide sequence of the product gel amplified by PCR is determined after the product gel is recovered, the determined nucleotide sequence is compared with the N-terminal sequence of the phage random dodecapeptide-gIII fusion protein, the amino acid sequence of the phage specifically bound with the target molecule can be deduced by referring to a degenerate codon table, and three polypeptides (A peptide (SEQ ID NO.1), D peptide (SEQ ID NO.2) and W peptide (SEQ ID NO.3)) with better binding property (Table 2) are selected to artificially synthesize the polypeptide with the purity of 95 percent.

TABLE 2 sequencing of phage binding clones

2.4 detection of polypeptide affinity

2.4.1 ELISA method for detecting the binding condition of affinity phage and EtMIC3-BC1

The titer was 1.0X 10 respectively⁶、1.0×10⁸、1.0×10¹⁰、1.0×10¹²The phage supernatant corresponding to the A, D, W peptide of pfu was reacted with EtMIC3-BC1(0.1mg/mL) recombinant protein and the binding of the selected polypeptide to EtMIC3-BC1 recombinant protein was examined. The results show that the phage supernatants corresponding to the three selected polypeptides can have certain reactivity with the EtMIC3-BC1 recombinant protein, and the binding capacity is enhanced along with the increase of the titer of the supernatants; in contrast, the binding capacity of the unrelated phage (VSV G protein affinity phage) to the EtMIC3-BC1 recombinant protein was not significantly altered. The results showed that three positive phages selected from the fourth round of bioscreening products were able to specifically bind to the EtMIC3-BC1 recombinant protein (FIG. 8).

2.4.2 binding of EtMIC3-BC1 Multi-antibody competitive inhibition affinity phage to EtMIC3-BC1 recombinant protein

FIG. 9 shows that rabbit anti-EtMIC 3-BC1 polyclonal antibody can inhibit to some extent the binding of the corresponding phage of the three polypeptides (A peptide, D peptide, W peptide) to the EtMIC3-BC1 recombinant protein, compared to unrelated polyclonal antibodies. The inhibition of the binding of the corresponding phages for peptide A and peptide D to the EtMIC3-BC1 recombinant protein increased with increasing antibody concentration, and the overall trend was enhanced. There was no significant change in the inhibitory effect of the addition of the unrelated polyclonal antibody. The three selected phage clones (especially the phage corresponding to the A peptide and the D peptide) are possible to simulate the binding epitope on rabbit anti-EtMIC 3-BC1 polyclonal antibody, and are well combined with EtMIC3-BC1 and have higher affinity.

2.4.3 ELISA detection of binding of Pest EtMIC3 protein to affinity phage

The binding of the three phage clones screened and the sporozoite sonicator was tested by ELISA. The results show (fig. 10) that three phage clones were able to bind to some extent to the sporozoite sonicator, with A, D peptide-corresponding phage binding to the sporozoite sonicator stronger than the W peptide-corresponding phage, while the unrelated phage (VSV G protein affinity phage) did not react significantly with the sporozoite sonicator, which demonstrates that the three selected phage were able to bind specifically to the etimic 3 protein.

Test example 2 in vitro test for the ability of polypeptide A, D, W to inhibit the invasion of cells by E.tenella sporozoites

1. Test method

1.1 MTT assay to determine the maximum non-toxic concentration of synthetic polypeptide on MDBK cells

(1) MDBK cells were prepared.

(2) Detecting the maximum nontoxic concentration of the polypeptide: MDBK cells in good growth status were plated in 96-well plates at 1.0X 10 per well⁴For each cell, the 96-well plate was placed at 37 ℃ with 5% CO₂Culturing in an incubator. Polypeptide A, D, W was prepared at 2mg/mL in serum-free DMEM cell culture medium, followed by dilution in multiples. After observing the growth of the cell monolayer to the bottom of the well, the supernatant culture solution is discarded, the well is washed 3 times by PBS, 1mg/mL, 0.5mg/mL, 0.25mg/mL and 0.125mg/mL of the polypeptide A, D, W are added into each well, 100 mu L of the polypeptide is added into each well, five duplicate wells are arranged for each concentration gradient of the three polypeptides, and a culture solution control well (only DMEM culture solution in each well) and a cell control well (only cells are paved in each well and no polypeptide is added) are arranged at the same time. The 96-well plates were further incubated at 37 ℃ with 5% CO₂The incubator lasts for 12 h. Add 10. mu.L of 5mg/mL MTT to each experimental well and continue the plates in the dark in CO₂The incubator lasts for 4 h. The culture medium in the wells was aspirated as much as possible, 150. mu.L of DMSO was added to each well, the mixture was allowed to stand at room temperature for 10min, and the mixture was shaken with a shaker for 10 min. The plate reader measures the OD at 490nm of the cell plate.

1.2 Collection, sporulation of oocysts and purification of sporozoites

Sporulation and collection of tenella oocysts:

(1) continuously collecting feces 7-11 days after chicken is infected with E.tenella, adding a proper amount of distilled water, stirring, soaking for about 3.5 hours, and stirring once every half hour, wherein the proper amount of distilled water is preferably used for stirring, and no obvious feces agglomeration exists; (2) filtering the soaked excrement by using a nylon sieve, subpackaging the obtained filtrate in a 50mL centrifuge tube, and centrifuging at 4500rpm for 10 min; (3) discarding supernatant, suspending the precipitate with saturated saline solution, mixing well, and centrifuging at 4500rpm for 10 min; (4) transferring the supernatant to another clean sterile centrifuge tube and adding distilled water with about one volume time of the supernatant, and centrifuging at 4500rpm for 10 min; (5) removing supernatant, completely suspending the precipitate with 2.5% potassium dichromate, mixing, and spreading on multiple plates; (6) and (3) culturing the flat plate in an incubator at 28 ℃, ensuring the humidity in the incubator to be proper, taking out the flat plate every 3-4 hours, blowing the flat plate once, and supplementing liquid to the flat plate with fast liquid volatilization. The oocyst suspension was observed under a microscope, and when the number of sporulated oocysts exceeded 80%, the sporulated oocysts were counted and collected and stored in a refrigerator at 4 ℃. E, collection and purification of Tenella sporozoites: the purification process of sporozoites needs aseptic operation, and equipment and reagents used for purification need aseptic treatment.

1.3 fluorescent labeling of E.tenella sporozoites

The purified and collected sporozoites are fluorescently labeled with a CFDA-SE cell proliferation and tracking detection kit according to the method of Huangyuchen et al.

1.4 detection of the ability of polypeptide A, D, W to inhibit sporozoites from invading MDBK cells

(1) Prior to assay, wells of 24-well cell culture plates were seeded with MDBK cells at 3X 10 per well⁴Individual cells, plates were incubated at 37 ℃ 5% CO₂In the cell culture chamber of (1), waitThe cell monolayer grows over the bottom of the wells of the culture plate; (2) the fluorescently labeled sporozoites are diluted with DMEM added with 1% double antibody and 10% serum and subpackaged, and 30 ten thousand sporozoites are subpackaged for each tube. Then 100. mu.L of polypeptide A, D, W at concentrations of 25. mu.g/mL, 50. mu.g/mL, 75. mu.g/mL, 100. mu.g/mL and 125. mu.g/mL, 1 tube per concentration of each polypeptide, were added to each tube. Meanwhile, 100. mu.L of rabbit anti-EtMIC 3-BC1 polyclonal antibody with the concentration of 100. mu.g/mL, 150. mu.g/mL and 300. mu.g/mL is respectively added into the other three tubes as a control for inhibiting the invasion of sporozoite, and polypeptide and polyclonal antibody are not added into the other single tube as an invasion control. And blowing and beating the solution in the tube uniformly, and setting 2 compound holes on each detection sample. The cell culture plates were placed at 37 ℃ in 5% CO₂Incubating for 2h in an incubator; (3) taking out the centrifuge tube, centrifuging at 1000rpm for 20min, carefully discarding the supernatant, and suspending the precipitate with 100 μ L cell culture solution; (4) the above suspension was added to 24 well cell culture plates, with 3 replicates per set. Incubate the plates again in CO₂An incubator is used for 10 h; (5) the plate was removed, the supernatant discarded, the cells on the plate were digested with 0.25% trypsin and gently blown up, the cell suspension was then transferred to a sterile 1.5mL EP tube, centrifuged at 1000rpm for 10min, and the supernatant aspirated. Washing the precipitate with PBS once, then suspending the cells in 500 mu L PBS, and keeping away from light at 4 ℃ for detection; (6) detecting the cell suspension in each EP tube by a flow cytometer, and calculating the polypeptide inhibition rate according to an invasion inhibition rate formula, wherein the calculation formula is as follows:

the invasion rate is the number of invading cells/(number of invading cells + number of uninjured cells) × 100%

The invasion inhibition rate is [1- (invasion rate embraced by polypeptide/invasion rate of sporozoite without polypeptide action) ] × 100%.

2. Test results

Purification of Tenella sporozoites and fluorescent labeling thereof

Mature oocysts obtained by collecting and purifying chicken droppings infected with E.tenella sporulated oocysts 7-11 d are sporulated, and sporozoites are purified by means of physical grinding, chemical digestion and the like (FIG. 11A). The purified sporozoites were fluorescently labeled according to the instructions of the FDA-SE test kit (FIG. 11B).

2.2 determination of the concentration of the polypeptide

After the A peptide, the D peptide and the W peptide with different concentrations are respectively acted on MDBK cells for 10 hours, and then the absorbance value at 490nm of each peptide is detected by a microplate reader. The results showed that the differences between the OD490nm of the MDBK cells and the cell control wells were all very significant (P < 0.01) when the concentrations of the A peptide (FIG. 12A), the D peptide (FIG. 12B) and the W peptide (FIG. 12C) were 1mg/mL, 0.5mg/mL, and 0.25mg/mL, and that the OD490nm of the MDBK cells was not significantly different from the cell control wells when the concentrations of the A peptide, the D peptide and the W peptide were 0.125mg/mL, indicating that the maximum non-toxic concentration of the A peptide, the D peptide and the W peptide to the MDBK cells was 125. mu.g/mL. In the figure, indicates that the difference is extremely significant (P < 0.01); indicates significant difference (P < 0.05). The notations of the images in the following figures are synonymous.

2.3 EtMC3-BC polyclonal antibody inhibits E.tenella sporozoite from invading MDBK cells

The ability of anti-EtMIC 3-BC1 polyclonal antibodies after 100-fold, 200-fold and 300-fold dilution to inhibit the invasion of E.tenella sporozoites into MDBK cells was examined by flow cytometry. The results showed that the inhibition rate of polyclonal antibody after 100-fold dilution for inhibiting e.tenella sporozoites from invading MDBK cells was 75.2%, which was significantly higher than the inhibition efficiency of polyclonal antibody after 200-fold and 300-fold dilution (P < 0.01) for e.tenella sporozoites from invading MDBK cells (fig. 13), thus serving as an antibody control after 100-fold dilution of polyclonal antibody at the original concentration when the inhibitory ability of polypeptide on e.tenella sporozoites from invading MDBK cells was examined.

2.4 inhibition of E.tenella sporozoites invading MDBK cells by Polypeptides

As shown in FIG. 14, the inhibition of invasion of the MDBK cells in vitro by the sporozoites in the group of peptides A, the group of peptides D and the group of peptides W were compared, and the remaining concentrations of all three polypeptides were significantly lower than those in the group of antibodies (P < 0.01), except that the A peptide was significantly lower than that in the group of antibodies (P < 0.05) at a concentration of 125. mu.g/mL. The inhibition rate of each concentration of the A peptide group is remarkably higher than that of the D peptide group and the W peptide group (P is less than 0.01); the inhibition rate of each concentration of the D peptide group is very obvious (P is less than 0.01) and is higher than that of the W peptide group. The inhibition rates of the A peptide, the D peptide and the W peptide on the invasion of E.tenella sporozoites into MDBK cells at a concentration of 125. mu.g/mL were 71.8%, 54.6% and 20.8%, respectively. The inhibition rates of the A peptide, D peptide and W peptide on the invasion of MDBK cells by E.tenella sporozoites were 39.2%, 20.4% and 4.8%, respectively, at a concentration of 25. mu.g/mL. The rate of inhibition of invasion of each polypeptide increased with increasing concentration of the polypeptide. Therefore, the screened three polypeptides can inhibit E.tenella sporozoites from invading MDBK cells to a certain extent, but the inhibition effect of the W peptide is poor, the effects of the A peptide and the D peptide are obviously superior to those of the W peptide, and the effect of the A peptide is superior to that of the D peptide.

Test example 3 in vivo test for detecting the protective action of the polypeptide against Positive phage coccidiosis

1. Test method

1.1 grouping and taking of experimental animals

75 good-standing 1-day-old laying hens were purchased from an incubator, scattered in a cleaned and sterilized incubator, and freely fed and drunk with food and water containing no anticoccidial drugs. When the chickens grew to 21 days of age, the chickens were randomly divided into 5 groups of 15, PBS, a, D, W, e. Infection and immunization procedures are shown in table 3:

TABLE 3 animal Experimental procedures

1.2 detection of weight gain Effect after insect attack

Weighing the weights of the chickens before attack and before killing the insects, and calculating the average increase and the relative weight gain rate according to a formula. The formula is as follows:

average increase (g) is pre-slaughter weight-pre-attack weight

Relative weight gain (%) - (pre-killer weight-pre-attacker weight)/PBS group increase;

1.3 grams fecal oocyst output count (OPG) and oocyst reduction rate

And on the seventh day after insect attack, collecting 3 parts of excrement, each part of excrement is 1g, soaking the excrement in deionized water and stirring the excrement and the excrement into paste, filtering the excrement by using gauze, transferring filtrate into a centrifugal tube, centrifuging the filtrate at 5000rpm for 10min, removing supernatant, suspending the precipitate by using a proper amount of saturated saline solution, centrifuging the precipitate at 3000rpm for 10min, collecting supernatant into another centrifugal tube, adding deionized water with the volume at least 2 times that of the centrifugal tube into the centrifugal tube, blowing and uniformly mixing the deionized water, centrifuging the mixture again, discarding the supernatant, fully suspending the precipitate by using PBS, diluting the precipitate, counting the diluted suspension by using a microscope, and calculating the oocyst discharge reduction rate according to the average value of three samples and a formula. The calculation formula is as follows:

OPG ═ a × 1/(0.1 × 0.1 × 0.1) × 10 (dilution factor) ═ a × 10⁵

Oocyst excretion reduction rate (%) (number of oocysts attacked by insect group-number of oocysts in each group)/number of oocysts attacked by insect group X100%

1.4 Scoring of caecum lesions

And (3) dissecting and killing all the chickens in each group on the eighth day after insect attack, observing pathological changes of the caecum, scoring according to a caecum lesion scoring standard, and counting 4 dead chickens according to the caecum with serious lesion serving as the standard in the scoring process. The scoring criteria were as follows:

0 minute: the cecum has normal eye sight and good tissue form;

1 minute: a few bleeding points can be observed on the wall of the cecum, the thickness is normal, and abnormal contents do not appear in the intestinal tract;

and 2, dividing: more obvious bleeding points can be observed on the wall of the cecum, the intestinal wall has slight swelling phenomenon, some lesions even appear, and a small amount of red excrement can be observed in the intestinal tract;

and 3, dividing: the cecal wall swelling is obvious, and the cecal core phenomenon is even generated in the intestinal tract;

and 4, dividing: the surface of the caecum wall is tense and extremely swollen, the intestinal wall is thickened, the atrophy phenomenon is obvious, and blood clots or white cheese substances can be observed in the intestinal tract.

1.5 anticoccidial index (ACI)

ACI is calculated according to the following calculation formula:

ACI ═ relative rate of weight gain + survival rate) - (oocyst value + lesion value)

Relative weight gain (weight gain of each group/weight gain of PBS group) × 100%

Survival rate (survival number of chickens in each group before killing/number of chickens in each group before attacking) x 100%

Mean lesion score (0-4) × 10 for each group

Oocyst values were converted as shown in Table 4.

TABLE 4 oocyst number conversion criteria

1.6 pathological histology of the cecum

Cecal tissues of all experimental groups of chickens were fixed by 10% formalin until the tissues became white, paraffin sections were prepared, and pathological changes of the tissues were observed by sectioning.

2. Test results

2.1 weight gain Change

The results showed that no mortality occurred and 100% survival occurred in each experimental group. The average body weight of the chickens in the PBS group is higher than that of the chickens in the other groups, compared with the attack control group, the average body weight of the chickens in the phage immune group corresponding to the A, D, W polypeptide is obvious (P < 0.05) and higher than that of the chickens in the E.tenella attack control group, and no statistical difference exists among the three phage immune groups (Table 5). The different capital superscripts indicate very significant differences between groups (P < 0.01), the lower case superscripts indicate significant differences (P < 0.05), and FIG. 15 is the same.

TABLE 5 weight change of chicks of each group

2.2 Coccidia infection Blind lesion score

After one week of E.tenella infection, the chickens in each experimental group did not die, and the caecum lesion scores of the chickens in groups A, D and W orally administered with the phages were significantly reduced compared with the control group of attack worms (P < 0.01). And the cecal lesion scores of group a and group D were significantly lower than those of group W; group a was not significantly different from group D. Shows that the oral phage has certain effect of resisting coccidian infection.

2.3 reduction rate of oocyst shedding

The number of oocysts purified from feces collected on day seven after infection with e.tenella in each group was shown (fig. 16), and the oocyst excretion reduction rate was 100% in the PBS group, 47.42% in the a group, 40.78% in the D group, and 8.52% in the W group, compared to the e.tenella attack insect control group, which had an oocyst excretion reduction rate of 0%. The result shows that the body can have a certain coccidian infection resistance effect by orally taking the bacteriophage.

2.4 anticoccidial index (ACI)

The ACI of each experimental group is calculated to be 200, 168.74, 161.27 and 118.21 respectively, wherein the ACI of the group A and the group D is more than 160, and the group W has a good protection effect on organisms and a certain anti-coccidiosis protection effect.

TABLE 6 anticoccidial index

Note: ACI is excellent above 180, good between 160 and 180, poor between 120 and 160, and ineffective below 120.

2.5 caecum pathological changes

In the test process, the E.tenella attack control group had diarrhea on day 3 after being infected with E.tenella sporulated oocysts, and the subsequent A, D and W groups had loose stools successively, so that the chickens in each group had normal mental status, normal food intake and drinking water. Except the PBS group, bloody stool appears in other experimental groups at the fifth day after insect attack, the insect attack control is most obvious, and the A group, the D group and the W group are slight. Chickens of the experimental groups were dissected at day 8 after infestation and observed for the pathological changes of the cecum eye. The results show (fig. 17) that the cecum in the PBS group (fig. 17, 1 and 2) was morphologically normal with no apparent macroscopic pathological changes. In comparison to the PBS group, the cecum was slightly swollen in groups a (fig. 17, 3 and 4) and D (fig. 17, 5 and 6), with scattered needle-size bleeding spots visible on the surface; cecum swelling was more evident in group W (fig. 17, 7 and 8), with increased superficial bleeding spots; the caecum on both sides of the control group (FIGS. 17, 9 and 10) was significantly congested and swollen, the intestinal wall was significantly thickened, and several bleeding spots with millet size were observed on the surface of the caecum.

As can be seen from the paraffin sections of the cecal tissues, the villi structure of the cecal tissues of the PBS group is relatively complete (FIG. 18A), and no obvious histopathological change can be observed due to the regular arrangement. The cecum tissue structure was relatively intact in group a (fig. 18B) compared to the PBS group, with a small number of red blood cells observable in the interstitial space; group D (FIG. 18C) the villus structure of the cecum tissue was slightly fragmented, and the interstitial space was scattered with red blood cells; group W (FIG. 18D) significant lesions appeared in the villus structure of cecum tissue, sloughed villus debris appeared in the intestinal lumen, and a large number of red blood cells were observed in the interstitial spaces; the cecal tissue structure of the chickens attacked the control group (fig. 18E) was seriously damaged, the intestinal villi fell in the intestinal cavity, and the tissue congestion phenomenon was serious.

SEQUENCE LISTING

<110> northeast university of agriculture

<120> chicken coccidium infection-resistant polypeptide and application thereof

<130> HLJ-1002-200508A

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 12

<212> PRT

<213> E. tenella

<400> 1

Ala Gly Arg Leu Leu Thr Pro Thr Met Ser Leu Val

1 5 10

<210> 2

<211> 12

<212> PRT

<213> E. tenella

<400> 2

Asp Tyr His Asp Pro Ser Leu Pro Thr Leu Gly Lys

1 5 10

<210> 3

<211> 12

<212> PRT

<213> E. tenella

<400> 3

Trp Lys Asp Val His Lys Ala Trp Leu Leu Glu Pro

1 5 10

Claims (8)

1. The polypeptide for resisting coccidium infection is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A gene encoding the polypeptide of claim 1.

3. An expression vector comprising the gene of claim 2.

4. The use of the polypeptide of claim 1 in the preparation of a medicament against coccidian infection in chickens; the coccidiosis is e.

5. A vaccine composition for preventing or treating coccidial infection in chicken, which comprises a prophylactically or therapeutically effective amount of the polypeptide of claim 1 and an immunoadjuvant; the coccidiosis is e.

6. The use of the gene of claim 2 in the preparation of a medicament against coccidian infection in chickens; the coccidiosis is e.

7. A DNA vaccine for preventing or treating coccidium infection in chickens, which consists of a prophylactically or therapeutically effective amount of the gene of claim 2 and an expression vector; the coccidiosis is e.

8. Use of the expression vector of claim 3 in the preparation of a medicament against coccidiosis in chickens; the coccidiosis is e.

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