Application of ribosomal proteins RPL27 and RPP0 in regulating drug resistance of chicken coccidia

Abstract

The present invention provides an application of chicken coccidia ribosomal proteins RPL27 and RPP0 in the regulation of chicken coccidia drug resistance. The results of this study show that the chicken coccidia ribosomal proteins RPL27 and RPP0 of the present invention can be used as a marker for detecting the drug resistance of chicken coccidia, and the pathogenicity of the insect strain after overexpressing RPL27 and RPP0 is significantly enhanced, and the drug resistance to maduramycin is enhanced. The results of this study provide an important basis for screening anticoccidial drug targets and vaccine molecules and screening chicken coccidia drug resistance molecular markers to establish a rapid method for detecting field coccidia drug resistance in the future, which is of great significance for green ecological healthy breeding and chicken coccidiosis prevention and control.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to application of chicken coccidia ribosomal proteins RPL27 and RPP0 in regulating the drug resistance of chicken coccidia. Background Art

[0002] Since the last century, chicken coccidiosis has caused huge economic losses to the poultry farming industry worldwide. This intracellular parasitic disease caused by the parasitism of Apicomplexa and Eimeria coccidia in different areas of the chicken intestine mainly manifests as damage to the host intestinal epithelial cells, resulting in poor absorption of nutrients, weight loss, mental depression, and intestinal bleeding in sick chickens, and even death in severe cases. At present, the prevention and control of chicken coccidiosis mainly relies on preventive medication or immune prevention with live oocyst vaccines (strong or weak vaccines), but at present, due to the many limitations of coccidiosis vaccines, such as high vaccine production costs, high logistics requirements, poor stability, short storage time, unsuitable for mass production or virulence reversal, etc., the widespread use of vaccines is limited, especially in the process of broiler breeding, so the prevention and control of chicken coccidiosis at this stage still mainly adopts drug prevention.

[0003] Anticoccidial drugs are mainly divided into two categories: 1) Chemically synthesized drugs are produced by chemical synthesis and have specific modes of action, including inhibiting the respiration of parasite mitochondria, the representative drug is decoquinate; inhibiting the folic acid synthesis pathway, the representative drug is sulfonamide; competitively inhibiting the uptake of thiamine, the representative drug is amprolium; drugs with other modes of action include diclazuril, halofuginone, nicarbazine, etc. The mechanism of action of diclazuril is generally believed to affect the synthesis of coccidian nucleic acid, block the normal growth of merozoites and microgametocytes, and thus lead to reproductive failure. 2) Polyether antibiotics or ion carriers, produced by fermentation of Streptomyces or Actinomycetes, these drugs destroy the ion gradient on the parasite cell membrane, mainly including monovalent ion carrier drugs, represented by salinomycin, methyl salinomycin, and monensin; divalent ion carriers, represented by lasalocid; monovalent glycoside ion carrier drugs, represented by maduramicin, which kills coccidia by destroying the normal intracellular ion balance of coccidia and affecting the activity of Na+-K+-ATPase. The consequences of long-term overdose of drugs are quickly manifested. Coccidia have developed varying degrees of resistance to almost all common drugs, and the situation has become increasingly serious and gradually developed into cross-resistance to multiple drugs. As the situation becomes more serious, scholars want to find out how coccidia resistance is generated in order to better control coccidiosis, but the mystery of the generation and development of chicken coccidia resistance has not yet been solved.

[0004] Ribosomal proteins play a vital role in ribosome assembly and protein translation. Studies on apicomplexan protozoa have found that Plasmodium falciparum ribosomal protein P2 is excreted onto the surface of invaded red blood cells during the early to mid-trophozoite stage. Artificial removal of P2 results in the inability of the parasite to undergo nuclear division (DAS et al., 2021). The ribosomal protein P2 of Toxoplasma gondii has also been shown to be associated with the invasion of parasites into host cells (SUDARSAN et al., 2015). In E. tenella, which is also an apicomplexan protozoan, Liang et al. (2020) found that the 60S large subunit ribosomal protein L12 (Et60s-RPL12) is involved in the invasion of coccidia into host cells and is located on the vacuole membrane of the parasite, indicating that in addition to constituting ribosomes and participating in protein biosynthesis, coccidia ribosomal proteins may also be involved in other functions such as parasite invasion and immune evasion. So far, there are no reports on the research of chicken coccidia ribosomal protein L27 and 60S large subunit acidic ribosomal phosphorylation protein P0, and the characteristics and functions of these two proteins are still unclear. Summary of the invention

[0005] In order to explore the exact mechanism of drug resistance in Eimeria tenella (E. tenella), our team used the drug concentration increment method in the early stage, using the E. tenella drug-sensitive strain (DS) isolated and preserved in the laboratory at the beginning of this century as the mother strain, and induced E. tenella diclazuril-resistant strain (DZR), maduramycin-resistant strain (MRR) and salinomycin-resistant strain (SMR) respectively. The obtained DZR is completely resistant to 1.2mg / kg diclazuril, MRR is completely resistant to 7.0mg / kg maduramycin, and SMR is completely resistant to 60mg / kg salinomycin, and the above-mentioned laboratory-induced resistant strains can be stably passaged.

[0006] In the early stage, the differentially expressed genes of DS, MRR and DZR were compared by transcriptome sequencing (RNA-sequencing, RNA-seq). Among them, the ribosomal protein L27 (ETH_00024495, EtRPL27) of E. tenella and the 60S large subunit acidic ribosomal phosphorylation protein P0 (ETH_00038080, EtRPP0) of Eimeria tenella were significantly different from those of DS in MRR. On this basis, the characteristics and functions of EtRPL27 and EtRPP0 were studied in this study.

[0007] Furthermore, the present invention provides an application of ribosomal protein L27 (ETH_00024495, EtRPL27) of E. tenella and 60S large subunit acidic ribosomal phosphorylation protein P0 (ETH_00038080, EtRPP0) of Eimeria tenella in the detection of drug resistance of Eimeria, wherein the drug resistance is preferably maduramycin resistance;

[0008] Furthermore, the present application provides a molecular marker that can quickly detect coccidia resistance, wherein the molecular marker is E. tenella ribosomal protein L27 (ETH_00024495, EtRPL27) and Eimeria tenella 60S large subunit acidic ribosomal phosphorylated protein P0 (ETH_00038080, EtRPP0).

[0009] Furthermore, the present application provides a use of a primer pair for respectively amplifying ribosomal protein L27 (ETH_00024495, EtRPL27) of Eimeria tenella and 60S large subunit acidic ribosomal phosphorylated protein P0 (ETH_00038080, EtRPP0) of Eimeria tenella in the preparation of a kit for rapid detection of molecular markers of coccidia resistance;

[0010] Preferably, the application is to use PCR detection to determine the resistance of chicken coccidia to maduramycin.

[0011] Furthermore, the primer pair is EtRPL27 (RPL27-F: 5'-ACAGGGACCTTTTGTTCTCG-3', RPL27-R: 5'-TCGTTTCCAGACCTTTGTCG-3'), EtRPP0 (RPP0-F: 5'-GGCGACAGTGCTGATGGGAAAG-3'; RPP0-R: 5'-CAGAAGATGAAGCCGACGTTGAGG-3').

[0012] Furthermore, the present invention provides a method for increasing the drug resistance of Eimeria tenella, the method comprising overexpressing ribosomal protein L27 and / or 60S large subunit acidic ribosomal phosphorylation protein P0 in E. tenella, wherein the drug resistance is preferably maduramycin resistance.

[0013] Beneficial Effects

[0014] EtRPL27 and EtRPP0 are not only involved in the normal function of E. tenella ribosomes - protein synthesis, but also in other important regulatory functions such as the invasion of host cells by the worm and the development of maduramycin resistance by the worm. The development of coccidia resistance, especially the emergence of multiple and cross-resistance, has caused many drugs to have poor anti-coccidia effects or even failure, and the development of new drugs is slow. Therefore, it leads to the abuse of drugs in chicken farms, causing animal bodies and their products to be polluted by drug residues, etc., and also destroys the environmental microecology, causing food safety hazards and threatening human health. Therefore, drug resistance has become the main obstacle in the current prevention and control of coccidiosis, and is an important obstacle to the healthy and sustainable development of the breeding industry. At present, the molecular mechanism of coccidia resistance is still unclear. The existing method for detecting chicken coccidia resistance is the chicken drug sensitivity test. This method can accurately detect the sensitivity of chicken coccidia to drugs, but it requires a certain number of chickens to be raised, which is expensive, has a long test cycle, is laborious, and requires well-trained experts to make judgments, which will largely lead to delays in on-site coccidiosis treatment. Therefore, there is an urgent need to establish a method for rapid and accurate detection of drug resistance in field chicken coccidia.

[0015] The results of the present invention provide a basis for analyzing the molecular mechanism of coccidia resistance to maduramycin and provide technical support for the establishment of a rapid molecular diagnosis method for coccidia resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The cDNA synthesized by reverse transcription of total RNA from sporozoites of sensitive strains of E. tenella was used as a template, and the products were detected by electrophoresis after PCR amplification. Wherein, M: DNA standard molecular weight (DL2000, 4500); Lane 1: PCR amplification product of EtRPL27; Lane 2: PCR amplification product of EtRPP0. The expected specific fragment length of EtRPL27 is 441bp, and the fragment length of EtRPP0 is 622bp.

[0017] Figure 2 Purified rEtRPL27 (A) and rEtRPP0 (B) were characterized by SDS-PAGE. M: protein standard; Lane 1: rEtRPL27; Lane 2: rEtRPL27.

[0018] Figure 3 The mRNA transcription levels of EtRPL27 and EtRPP0 in different developmental stages of susceptible strains of E. tenella. UO: unsporulated oocyst; SO: sporulated oocyst; SZ: sporozoite; SM: second generation merozoite.

[0019] Figure 4The mRNA transcription levels of EtRPL27 and EtRPP0 in different insect strains. DS: sensitive strain; DZR: diclazuril-resistant strain; MRR: maduramycin-resistant strain; SMR: salinomycin-resistant strain.

[0020] Figure 5 IFA detected the distribution of EtRPL27 in different stages of the worm. SZ: sporozoites incubated with PBS for 2 h; ~h pi: sporozoites invading DF-1 cells ~h; SM: second-generation merozoites incubated with PBS for 2 h. Scale bar = 10 μm;

[0021] Figure 6 IFA detected the distribution of EtRPP0 in different stages of the worm. SZ: sporozoites incubated with PBS for 2 h; ~h pi: sporozoites invading DF-1 cells for ~h; SM: second-generation merozoites incubated with PBS for 2 h. Scale bar = 10 μm;

[0022] Figure 7 Effects of rabbit anti-rEtRPL27 (A) and rEtRPP0 (B) polyclonal antibodies on sporozoite invasion of DF-1 cells in vitro.

[0023] Figure 8 Sporulated oocysts of E. tenella strains overexpressing RPL27 and RPP0 genes.

[0024] Fig. 9 Flag tag antibody was used to identify overexpressing insect strains by Western blot.

[0025] Fig.10 The results of in vitro invasion rate detection of sporozoites of overexpressing insect strains (EtRPL27-OE and EtRPP0-OE) and the mother strain DS. Example

[0026] Insect strains: The E. tenella sensitive strain (DS, resource number: CAAS21111601) used in this experiment was isolated by the Animal Parasite and Biological Vector Control Team of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences in 2001. It was found to be sensitive to multiple drugs through drug sensitivity tests in chickens, and it has been regularly passaged and preserved to date; the E. tenella diclazuril-resistant strain (DZR, resource number: CAAS21111608) and the E. tenella maduramycin-resistant strain (MRR, resource number: CAAS21111609) were induced by this laboratory in 2004, and the E. tenella salinomycin-resistant strain (SMR, resource number: CAAS21111618) was induced by this laboratory in 2019. DZR, MRR, and SMR have been regularly passaged and preserved to date; the EtHCYA strain used in the in vitro inhibition experiment was from the College of Veterinary Medicine, China Agricultural University, and was constructed, stably passaged, and donated by Professor Suo Xun's research group.

[0027] The chicken embryo fibroblasts (DF-1) and human embryonic kidney cells (HEK293T) used in the experiment were provided by the Animal Parasite and Biological Vector Control Team of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0028] The three-yellow chickens used in this experiment were purchased from Shanghai Minyou Poultry Farming Professional Cooperative. On the day of hatching, they were transported to the experimental animal room of the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, which had been thoroughly disinfected in advance, for isolation and breeding. The feed and drinking water were tested and confirmed to contain no anticoccidial drugs. The experimental chickens were raised under coccidia-free and antibiotic-free conditions. The feces of the experimental chickens were checked before the experiment to ensure that there was no coccidia infection before the experiment could be carried out.

[0029] The New Zealand white rabbits used for the preparation of polyclonal antibodies in the experiment were purchased from Songlian Experimental Animal Facility in Songjiang District, Shanghai, and the Balb / c mice were purchased from Sibefore (Beijing) Biotechnology Co., Ltd. All experimental animals were housed in a coccidia-free clean-grade animal room established by the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0030] Example 1 Amplification and expression of EtRPL27 and EtRPP0 genes

[0031] PCR amplification of EtRPL27 and EtRPP0 gene ORF

[0032] The sporulated oocysts of E. tenella were collected, and the total RNA of the sporulated oocysts was extracted using TRIzol reagent. The mRNA in the total RNA was reverse transcribed into the first strand of cDNA using Vazyme HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription reagent. The first strand of cDNA was used as a template to obtain the ORFs of the target genes EtRPL27 and EtRPP0 by PCR in vitro amplification. Specific primers were designed according to the ORFs of EtRPL27 and EtRPP0. The primers are as follows:

[0033] Table 1 PCR primer sequences

[0034]

[0035] Construction of prokaryotic expression recombinant plasmid and expression of recombinant protein

[0036] The amplified PCR products were detected by 1% agarose gel ( Figure 1 ), cut off the target band at the correct position, and purify and recover the target gene according to the gel recovery kit operation manual. The purified EtRPL27 and EtRPP0 target fragments were connected to the pGEM-T Easy vector and transformed into E. coli Tans1-T1 competent cells, IPTG and X-gal were added for blue-white screening, and white colonies were selected to LB (containing 0.1% ampicillin) liquid culture medium for 8 hours. After PCR identification of the bacterial solution, the bacterial sample with the correct band position was sent to a biological company for sequencing. The results of BLAST online tool analysis showed that the amplified sequences had a 100% similarity with the known E. tenella ribosomal protein L27 (GenBank: XM_013375230.1) and E. tenella 60S large subunit acidic ribosomal protein P0 (GenBank: XM_013374540.1) genes, proving that the target fragments EtRPL27 and EtRPP0 were successfully cloned.

[0037] The bacterial solution that was correctly identified by sequencing was used to extract the recombinant cloning plasmids pGEM-T-EtRPL27 and pGEM-T-EtRPP0 according to the instructions of the small plasmid extraction kit. The extracted recombinant cloning plasmids pGEM-T-EtRPL27 and pGEM-T-EtRPP0 and the prokaryotic expression vector pGEX-4T-1 (containing GST tag) were double-digested with restriction endonucleases EcoRI and XhoI, respectively, and recovered and ligated, and transformed into E. coli Trans1-T1 competent cells. After the bacterial solution was correctly identified by PCR and sequencing, the recombinant plasmids pGEX-4T-EtRPL27 and pGEX-4T-EtRPP0 were extracted and transformed into E. coli BL21 (DE3) competent cells, respectively. Single colonies were picked and cultured in 4 mL of LB (containing ampicillin) liquid culture medium at 37°C, 180 r / min for 12 hours; 4 mL of the cultured bacterial solution was taken and added to 200 mL LB liquid medium (resistance is the same as above), culture at a constant temperature of 37°C and 180r / min with shaking. When the OD600nm value of the bacterial solution measured by a UV spectrophotometer reaches above 0.7, add 20μL IPTG (final concentration 1mM) was induced for 12h; the bacterial solution was centrifuged at 4℃, 10000r / min for 10min, the precipitate was resuspended in 30mL of autoclaved PBS and ultrasonically broken on ice, ultrasonically for 5s and then stopped for 5s, the power was 30%, and the ultrasonic was performed for 20-30min; after the end of the ultrasonication, the supernatant and the precipitate were centrifuged at 4℃, 10000r / min for 10min, and the supernatant and the precipitate were detected by SDS-PAGE. Both rEtRPL27 and rEtRPP0 were expressed in the form of inclusion bodies. The rEtRPL27 and rEtRPP0 proteins were purified by gel excision recovery. The SDS-PAGE detection showed that the expected molecular weight of rEtRPL27 was 43.2kDa, and the rEtRPP0 band was about 49.8kDa, which was consistent with expectations. Figure 2 shown.

[0038] Example 2 Differential analysis of mRNA transcription levels of EtRPL27 and EtRPP0 in different developmental stages and different strains of E. tenella DS

[0039] Differential analysis of mRNA transcription levels at different developmental stages in susceptible strains of E. tenella

[0040] Unsporulated oocysts (UO), sporulated oocysts (SO), sporozoites (SZ) and second-generation merozoites (SM) of sensitive strains of E. tenella were collected and purified, and total RNA of the four developmental stages was extracted using Trizol reagent. The total RNA was reverse transcribed into the first-chain cDNA using Vazyme HiScriptIII RT SuperMix for qPCR (+gDNA wiper) reverse transcription reagent and random primers. qPCR detection was performed using ABi 7500 fast real-time fluorescence quantitative PCR system and TB Green dye with 18S rRNA as reference. The primer sequences are shown in Table 2, the experimental system is shown in Table 3, and the reaction conditions are shown in Table 4. The results are shown in Table 2. Figure 3 As shown: the mRNA transcription level of EtRPL27 was the highest in the SZ stage, followed by SM, and was the lowest in the UO and SO stages; the mRNA transcription level of EtRPP0 reached a peak in the UO stage, followed by SM, and was the lowest in the SZ and SO stages.

[0041] Table 2 qPCR primer list

[0042]

[0043] Table 3 Experimental system

[0044]

[0045] Table 4 Reaction conditions

[0046]

[0047]

[0048] Differential analysis of mRNA transcription levels of EtRPL27 and EtRPP0 in sensitive and resistant strains of E. tenella

[0049] To analyze the differences in the mRNA transcription levels of EtRPL27 and EtRPP0 in the sporozoite stage between E. tenella sensitive strain (DS) and three different resistant strains (diclazuril resistant strain (DZR), maduramycin resistant strain (MRR) and salinomycin resistant strain (SMR), this experiment used 18S as a reference and qPCR for detection. The specific operation steps were the same as above. The results are shown in Figure 4 As shown: The transcription levels of EtRPL27 and EtRPP0 were significantly upregulated in MRR (p<0.001), but there was no significant difference in DZR and SMR.

[0050] Example 3 IFA detection of the distribution and localization of EtRPL27 and EtRPP0 in different developmental stages of sensitive strains of E. tenella

[0051] 1) Preparation of polyclonal antibodies: For the first immunization, the recombinant proteins rEtRPL27 and rEtRPP0 were mixed and emulsified with the same volume of Freund's complete adjuvant (FCA) to immunize New Zealand white rabbits at 200 μg / rabbit and balb / c mice at 50 μg / rabbit. Two weeks after the first immunization, the recombinant protein was emulsified with Freund's incomplete adjuvant (FIA) for the second immunization. After the second immunization, two booster immunizations were performed every other week for a total of 4 immunizations. One week after the last immunization, serum (anti-rEtRPL27 and rEtRPP0 polyclonal antibodies) was collected and stored at -20°C for use.

[0052] 2) Place the cell slide in a six-well plate and add 2×10 5 DF-1 cells were inoculated into the slides in a six-well cell plate at a number of cells / well and cultured in a cell culture incubator containing 5% CO2 and 37°C for 16 hours.

[0053] 3) Collect freshly purified sporozoites at a rate of 6×10 per well. 5 The number of sporozoites added to the wells was 3:1, added to a six-well plate containing DF-1 cells, and cultured in a 37°C, 5% CO2 cell culture incubator to allow the sporozoites to invade the DF-1 cells and develop therein.

[0054] 4) After the sporozoites were added to DF-1 cells for 2h, 12h, 24h, 48h, 72h, and 84h, the cell slides in the six-well plate were taken out and PBS was added at 2mL / well to wash the cell slides 3 times for 5min each time to remove impurities such as floating cells after aging and death in the culture medium. At the same time, the freshly collected and purified sporozoites and second-generation merozoites were suspended in PBS and directly applied to the cell slides and air-dried.

[0055] 5) Use 2 mL of cell fixative (4% paraformaldehyde) to fix sporozoites, second-generation merozoites and parasites at different developmental stages of sporozoites invading DF-1 cells for about 25 minutes. Then place the six-well plate in a decolorizing shaker and wash with PBST at a constant speed for 3 times, each time for 5 minutes.

[0056] 6) After fixation, add 2 mL of 1% Triton X-100 and place at room temperature for 25 min. Wash with PBST three times, 5 min each time.

[0057] 7) Add 2% bovine serum albumin (BSA) blocking solution to the six-well cell plate at 2 mL / well and block at 4°C for 16 h. Wash the cells three times with PBS, each time for 5 min.

[0058] 8) Rabbit anti-rEtRPL27 and rEtRPP0 (1:300 dilution) polyclonal antibodies were added to the six-well cell plate, incubated in a 37°C incubator for 1 hour, and then washed three times with PBST, each time for 5 minutes.

[0059] 9) After the primary antibody incubation, add 1:500 diluted fluorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG to the six-well cell plate in a dark environment, incubate at 37°C for 50 minutes, and then wash with PBS three times, each time for 5 minutes. After the secondary antibody incubation, add DAPI diluted 1:1000, incubate at room temperature for 10 minutes, and wash with PBS three times, each time for 5 minutes.

[0060] 10) Take a clean glass slide and drop a small amount of anti-fluorescence quencher, carefully take out the cell slide from the six-well plate, turn it upside down on the glass slide to seal it, and place it under an upright fluorescence microscope to observe the distribution and localization of EtRPL27 and EtRPP0 in the worm body.

[0061] The localization results of EtRPL27 protein are shown in Figure 5 As shown, in the sporozoite stage of E. tenella, it is diffusely distributed in the cytoplasm and cell membrane of the insect body except for the refractozoite, and is dotted on the surface; in the second generation of merozoites, it is located in the cytoplasmic matrix of the insect body, and the fluorescence intensity is weak; as the sporozoites invade DF-1 cells and develop into trophozoites, the EtRPL27 protein is evenly located in the cytoplasm of the insect body and the fluorescence gradually increases, but after developing into mature schizonts, the fluorescence intensity weakens. No fluorescence was observed in the negative control.

[0062] The positioning results of EtRPP0 are as follows Figure 6 As shown, the protein is mainly distributed in the cytoplasm and surface of sporozoites. When sporozoites invade DF-1 cells, it is especially concentrated in the refracting body, a cell organelle unique to sporozoites. When the parasite undergoes schizogony in DF-1 cells, EtRPP0 is evenly distributed in the cytoplasm, with granular dot distribution and enhanced fluorescence intensity. In the second generation of merozoites, the protein is evenly distributed throughout the parasite cell matrix, and the fluorescence around the cell nucleus is brighter.

[0063] Example 4 Rabbit anti-rEtRPL27, rEtRPP0 polyclonal antibodies inhibit sporozoite invasion in vitro

[0064] The collected rabbit anti-rEtRPL27, rEtRPP0 polyclonal serum and the subsequent TBS buffer and glycine eluent (pH = 1.9, pH = 2.7) were sterilized by filtration with a 0.45 μm filter membrane, and IgG in the serum was purified using Protein A + G Agarose according to the instructions. SDS-PAGE electrophoresis was used to observe whether the IgG was intact, and its concentration was determined using a BCA kit and stored in a -20°C refrigerator.

[0065] 1) 2×10 5 DF-1 cells were inoculated at a number of per well and cultured in a constant temperature incubator at 37°C and 5% CO2 for 12 h.

[0066] 2) Fresh EtHCYA sporozoites were extracted and resuspended in 10% complete medium (containing 10% fetal bovine serum, 3% streptomycin + penicillin), and different IgG concentrations (50, 100, 200, 300, 400 μg / mL) were set up. IgG was added to the resuspended sporozoites to a final IgG concentration of 50, 100, 200, 300, 400 μg / mL, respectively. The sporozoites were then incubated at 37°C for 2 h. Healthy rabbit IgG and no IgG groups were set up as controls.

[0067] 3) Take out the incubated sporozoites, centrifuge at 3000r / min for 5min, discard the supernatant, and resuspend the sporozoites in an appropriate amount of complete medium. 5 The cells were added into a 24-well plate containing DF-1 cells and cultured in a cell culture incubator at 41°C and 5% CO2 for 10 h.

[0068] 4) Take out the DF-1 cells containing sporozoites and aspirate the culture medium. Add an appropriate amount of PBS to each well and wash twice to remove the residual culture medium and cell metabolites. Add an appropriate amount of trypsin digestion solution to digest the cells for 2 minutes. After the cells are completely detached from the wall of the culture plate, add an appropriate amount of complete culture medium to terminate the digestion. Collect all the cell suspension and transfer it to a 1.5 mL EP tube.

[0069] 5) After centrifugation at 2000 r / min for 7 min, add appropriate amount of PBS to wash the precipitate twice to remove excess impurities, add 300 μL PBS to resuspend the precipitate, and use flow cytometry to detect the invasion rate of sporozoites into DF-1 cells.

[0070] 6) The invasion inhibition rate was calculated according to the formula [invasion inhibition rate = (1-(invasion rate of sporozoites in the experimental group incubated with antibodies / invasion rate of sporozoites in the untreated control group) × 100%)]. (Three replicates were set for all variables; EtHCYA has its own green fluorescence, which can be directly detected by flow cytometry.)

[0071] The inhibitory effect of rabbit anti-rEtRPL27 IgG on sporozoite invasion in vitro Figure 7As shown in A: The inhibitory effect of specific antibody IgG is dose-dependent, and its inhibition rate is positively correlated with the concentration used within a certain concentration range. Since the antibody IgG concentration began to show significant differences from 100μg / mL, when the working concentration was 300μg / mL, the invasion inhibition rate of anti-rEtRPL27-IgG on sporozoites was about 40.67%, and when the concentration was in the range of 50μg / mL to 400μg / mL, the invasion inhibition rate was higher than that of the negative control healthy rabbit serum IgG group, and the difference was significant or even extremely significant.

[0072] The inhibition rate of rabbit anti-rEtRPP0 IgG on sporozoite invasion of DF-1 cells in vitro is as follows Figure 7 As shown in B: The inhibitory effect of specific rabbit anti-rEtRPP0-IgG is dose-related. The higher the IgG concentration used to incubate sporozoites, the lower the invasion rate. At 400 μg / mL, the inhibition rate reached 40.33%. When the concentration was in the range of 50 μg / mL to 400 μg / mL, the invasion inhibition rate was higher than that of the negative control healthy rabbit serum IgG group, and the difference was significant.

[0073] Example 5 Construction of EtRPL27 and EtRPP0 overexpression strains

[0074] Construction of overexpression recombinant plasmids (EtRPL27-OE and EtRPP0-OE)

[0075] 1) According to the ORF sequences of EtRPL27 and EtRPP0 genes, specific primers with homology arms of the overexpression vector MEM-OE (kindly donated by Professor Suo Xun, College of Veterinary Medicine, China Agricultural University) were designed: ORF fragments of EtRPL27 and EtRPP0 (EtRPL27, EtRPP0) were amplified from the correctly identified cloned recombinant plasmids pGEM-T-EtRPL27 and pGEM-T-EtRPP0; Mic (promoter part) fragment and 3'Actin-vector backbone fragment (fragment 3'Actin) were obtained by in vitro amplification from the overexpression vector MEM-OE. The primer sequence information used is shown in Table 5.

[0076] Table 5 PCR primer sequences for constructing overexpression strains

[0077]

[0078]

[0079] 2) Recover the target fragments: Detect the PCR results by agarose gel electrophoresis, cut out the target bands (EtRPL27, EtRPP0, Mic fragment and 3'Actin fragment) at the correct position and use a gel recovery kit to recover and purify the target bands. For specific operations, refer to the kit operation guide.

[0080] 3) Construction of overexpression recombinant plasmids (EtRPL27-OE and EtRPP0-OE): ClonExpress MultiS One Step Cloning Kit was used for multi-fragment ligation. According to the instructions, the fragments EtRPL27 and EtRPP0 were assembled onto the 3'-Actin fragment and the Mic fragment, respectively, to obtain the EtRPL27-OE and EtRPP0-OE overexpression recombinant plasmids.

[0081] 4) Transformation: The ligation system was transformed into E. coli Trans1-T1 competent cells and cultured in LB solid medium at 37°C for 15 h.

[0082] 5) Identification of positive clones: Single clones were picked and cultured in LB liquid medium for 10 h. PCR amplification was performed to identify whether they contained the target genes EtRPL27 and EtRPP0. The positive clone strains containing the target fragments (EtRPL27 and EtRPP0) were sent to Bio-Tech for sequencing to detect whether the plasmid was successfully constructed.

[0083] Overexpression recombinant plasmids (EtRPL27-OE and EtRPP0-OE) were transfected into E. tenella

[0084] 1) Linearization of overexpression recombinant plasmid: 4 mL of the monoclonal bacterial solution containing the overexpression recombinant plasmid (EtRPL27-OE and EtRPP0-OE) was taken and cultured in 200 mL LB liquid medium for 16 h to extract a large amount of recombinant plasmid. The extracted recombinant plasmid was linearized by SnaBI restriction enzyme digestion.

[0085] 2) Purify the linearized plasmid: add an equal volume of DNA extract to the above SnaBI digested system, gently invert and mix, let stand at room temperature for 5 minutes, and centrifuge at 4°C, 12000r / min for 8 minutes; transfer the upper clear liquid after centrifugation to a new 1.5mL centrifuge tube, add pre-cooled isopropanol equal to the liquid in the tube, transfer to -20°C and let stand for 30 minutes, centrifuge at 4°C, 12000r / min for 15 minutes to precipitate the linearized recombinant plasmid, discard the supernatant, wash the precipitate at the bottom of the tube with pre-cooled 75% ethanol, centrifuge, repeat twice; let stand at room temperature to air dry the residual alcohol in the tube, leave for about 5 minutes, add 50μL RNase-free ddH2O after the ethanol evaporates, and repeatedly pipette until the linearized plasmid DNA is completely dissolved. After measuring the concentration, set aside.

[0086] 3) Transfection of E. tenella sensitive strain sporozoites with overexpressed recombinant plasmid: Purified E. tenella sensitive strain sporozoites were collected, reagents were added according to the system in Table 6, and the mixture was gently pipetted for use. The transfection reaction system is shown in Table 7.

[0087] Table 6 Reagents and volumes required for a single reaction

[0088]

[0089]

[0090] Table 7 General system for transfection of Eimeria sporozoites

[0091]

[0092] The LONZA4D-Nucleofecter instrument was used to perform transfection according to the instructions of the manual, and the EO-114 program was selected. Immediately after the transfection, 200 μL of DMEM medium preheated to room temperature was added to the nuclear transfer cup to protect the sporozoites. The transfected sporozoites were then inoculated into 7-day-old coccidia-free chicks, which were fasted for 12 hours before inoculation, and 5×10 per chick. 6 Sporozoites were inoculated through the cloaca. Water and feed were given 10 hours after inoculation, and 150 mg / kg pyrimethamine was added to water and feed during the culture period.

[0093] Screening and enrichment of EtRPL27-OE and EtRPP0-OE overexpression positive strains

[0094] Flow cytometry and pyrimethamine were used to enrich EtRPL27-OE and EtRPP0-OE overexpression positive strains.

[0095] The oocysts transfected with the overexpression recombinant plasmid (EtRPL27-OE / EtRPP0-OE) were collected and purified after sporulation reached 90%. + ) were collected in flow cytometry tubes and then counted at 1.0×10 3 The sporulated oocysts / chicken were orally inoculated into 5-day-old chicks, and the chicks were fed with water and feed supplemented with 300 mg / kg pyrimethamine. The above flow screening and drug passage enrichment were repeated 2-3 times until the green fluorescent insect bodies in the population reached more than 50%.

[0096] Isolation of single oocysts from EtRPL27-OE and EtRPP0-OE overexpression positive strains

[0097] In order to obtain a relatively pure overexpression strain (EtRPL27-OE / EtRPP0-OE), the mixed population of oocysts containing the EtRPL27-OE / EtRPP0-OE overexpression strain enriched by flow cytometry and drugs were rejuvenated and then single oocysts were isolated. The specific steps are as follows:

[0098] 1) Sporulated oocysts stored in 2.5% potassium dichromate were centrifuged at 3600 r / m for 5 min to remove the potassium dichromate, and washed twice with tap water, and then resuspended with an appropriate amount of pure water for later use.

[0099] 2) Weigh 1.3 g agarose into a clean beaker (bake at 80°C for 15 min before use), add 70 mL ddH2O, and heat in a microwave oven to fully dissolve.

[0100] 3) Pipette the dissolved agarose and spread it on a new slide, wait for it to cool and solidify, and cut it into 0.5×0.5cm pieces with a blade. 2 Reserve small pieces for later use.

[0101] 4) Place the cut agarose gel block under a fluorescence microscope, adjust the focus, use a toothpick to pick up the oocyst suspension containing the EtRPL27-OE / EtRPP0-OE overexpressing strain and lightly tap it on the gel block, and observe under a fluorescence microscope. When there is only one sporulated oocyst on the entire gel block and all four sporangia emit green light, feed the gel block to chicks within 3 days of age.

[0102] 5) One week after inoculation of a single oocyst, check whether there are oocysts in the feces of individual chicks. If there are oocysts in the feces, kill the chicken, take the cecum, collect the oocysts and sporulate them in 2.5% potassium dichromate, which is the homozygous EtRPL27-OE / EtRPP0-OE overexpression strain. After breeding in chickens, collect the sporulated oocysts and store them in 2.5% potassium dichromate at 4°C for later use.

[0103] Under a fluorescence microscope, it was observed that the original non-luminescent strain (mother strain) had oocysts expressing YFP+ green fluorescence, indicating that the linearized plasmid containing the EtRPL27-OE / EtRPP0-OE fragment was successfully integrated into the E. tenella genome. After multiple sorting by flow cytometry and enrichment with pyrimethamine until the positive rate was higher than 50%, single oocysts were isolated under a fluorescence microscope and inoculated into chicks, and two homozygous overexpression strains (EtRPL27-OE and EtRPP0-OE) were obtained. Figure 8 shown.

[0104] Identification of EtRPL27-OE and EtRPP0-OE overexpressing strains

[0105] The whole protein of sporulated oocysts of EtRPL27-OE and EtRPP0-OE overexpressing strains was extracted and then detected by Western blot. 2.0×10 7Each tube was added with an equal volume of glass beads, 400 μL RIPA (strong) lysis buffer and 1% protease inhibitors, and the oocyst wall was broken by vortexing at 2500 r / min for a total of 30 minutes. Then ultrasonically broken for 5 minutes, working for 2 seconds and stopping for 2 seconds. Centrifuge at 4°C, 12000 r / min for 10 minutes, carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube to determine the protein concentration. Western blot was performed using the Flag tag antibody as the primary antibody to identify the EtRPL27-OE and EtRPP0-OE overexpressing strains. The results are shown in Fig. 9 As shown: EtRPL27-OE and EtRPP0-OE strains have bands at 24.3 kDa and 18.7 kDa, respectively, indicating that the overexpression strains of these two genes were successfully constructed.

[0106] In vitro invasion assay of EtRPL27-OE and EtRPP0-OE overexpressing strains

[0107] One day before the sporozoite invasion, 2.0×10 5 The cells were inoculated into 24-well plates in the amount of 100 μg / well. The cell culture medium was complete medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin + streptomycin). The cells were cultured in an incubator at 37°C and 5% CO2. Sporozoites of sensitive strains of maternal insect strains DS, EtRPL27-OE, and EtRPP0-OE overexpressing strains were purified. The CFDA-SE cell tracing kit was used to fluorescently label the sporozoites of the sensitive strains of the maternal insect strain according to the instructions. Since the overexpressed insect strains spontaneously fluoresce, the same processing steps were performed. The above-incubated sporozoites were respectively divided into 6.0×10 5 / well to invade DF-1 cells, and incubate at 41℃, 5% CO2 for 10h, then wash the cells once with PBS. Add 0.25% Trypsin at 200μL / well to digest the cells, and add 300μL complete medium after 2min to terminate the digestion. Transfer the cell suspension to a 1.5mL centrifuge tube, centrifuge at 1000r / min for 5min, and carefully discard the supernatant. Wash the precipitate twice with 1×PBS. Finally, add 300μL PBS and transfer to the flow cytometry loading tube, and use flow cytometry to detect the invasion rate of sporozoites.

[0108] The results are as follows Fig.10 As shown: Compared with the sensitive strain of the maternal insect strain, the sporozoite invasion rate of the EtRPL27-OE and EtRPP0-OE overexpressing insect strains increased, indicating that the increase in the expression of EtRPL27 and EtRPP0 genes increased the invasion of host cells. Example 6 Animal clinical trials of EtRPL27-OE and EtRPP0-OE overexpressing insect strains

[0109] In vivo pathogenicity detection of EtRPL27-OE and EtRPP0-OE overexpressing insect strains

[0110] Ten-day-old chicks with similar body weight were selected, 6 per group, and divided into 10 groups on average. Except for the uninfected group, the other groups were orally infected with sporulated oocysts of the maternal parasite sensitive strain (DS), EtRPL27-OE and EtRPP0-OE overexpressing strains. Different attack doses were set according to the concentration gradient. All experimental chickens were killed on the 8th day after infection. The specific groups and attack doses and their results are shown in Table 8.

[0111] Average weight gain, OPG (oocysts per gram of feces), lesion score and mortality were used as experimental indicators, and the weight gain values ​​were statistically processed; the lesion score was based on the established standard (JOHNSON et al, 1970), and all chickens that died of coccidia in the experiment had cecal lesions scored as 4 points. The DS of the EtRPL27-OE / EtRPP0-OE overexpression strain and the maternal strain were compared, and the weight gain, oocyst output and lesion degree of each group were statistically analyzed to evaluate the virulence changes of the overexpression strain.

[0112] Table 8 Pathogenicity determination experimental groups and results

[0113]

[0114] Note: Same shoulder marks indicate no significant difference, different shoulder marks indicate significant difference. WGR, weight gain rate.

[0115] The results showed that the weight gain of chicks infected with different doses of EtRPL27-OE and EtRPP0-OE overexpression strains was lower than that of the infected maternal strain (DS). The higher the attack dose, the less weight the infected animals gained. Among the groups infected with overexpression strains, the oocyst production of the low- and medium-dose infection groups was higher than that of the DS group, while the oocyst production of the high-dose attack group was lower than that of the DS group, which may be related to the "crowding effect" of the parasitic site. There was no significant difference in the cecal lesion scores between the overexpression strain group and the DS group, indicating that the damage of the overexpression strain to the intestinal tract of chicks was similar to that of the maternal strain. In vitro and in vivo tests found that compared with the maternal strain DS, the two overexpression strains had an increased invasion rate of cells in vitro and enhanced pathogenicity to chicks.

[0116] Determination of drug resistance of EtRPL27-OE and EtRPP0-OE overexpressing insect strains

[0117] In vivo drug resistance testing group

[0118] The drug-treated group of the overexpressed strain (EtRPL27-OE / EtRPP0-OE) was compared with the maternal strain (DS) to evaluate whether the overexpressed strain was resistant to maduramycin. Ten-day-old chicks with similar body weight were selected, with 6 chicks in each group. The infected group was fed with maduramycin-containing feed from the day of grouping until the end of the experiment. One day after grouping, each chicken in each infected group except the healthy control group was orally inoculated with 5.0×10 5 sporulated oocysts. The specific grouping, drug feeding and results are shown in Table 9. On the 6th to 8th day after infection, feces were collected every day to calculate OPG; after the 8th day of infection, all experimental chickens were weighed and killed, and the weight gain rate of each group was calculated. At the same time, the cecal lesions were scored according to the standard of Johnson et al. (1970). All chickens that died of coccidia in the experiment had cecal lesions scored as 4 points. Referring to the method introduced by Huang Bing et al. (2001), the coccidia resistance was comprehensively evaluated with four indicators: anticoccidial index (ACI), lesion score reduction rate (RLS), relative oocyst production (ROP) and optimal anticoccidial activity percentage (POAA). If all four indicators are negative, it is not resistant; if one indicator is positive, it is mildly resistant; if two indicators are positive, it is moderately resistant; if 3-4 indicators are positive, it is completely resistant.

[0119] Table 9 Grouping and results of drug resistance determination experiment

[0120]

[0121] The results showed that the EtRPL27-OE strain was completely resistant to 2 mg / kg maduramycin and moderately resistant to 3 mg / kg maduramycin; the EtRPP0-OE strain was completely resistant to 2 mg / kg maduramycin and slightly resistant to 3 mg / kg maduramycin. MRR was completely resistant to 5 mg / kg maduramycin.

[0122] Based on the above results, we can see that the expression of E. tenella ribosomal protein L27 (EtRPL27) and Eimeria tenella 60S large subunit acidic ribosomal phosphorylation protein P0 (EtRPP0) in E. tenella maduramycin-resistant strains is significantly higher than that in sensitive strains and other resistant strains. Overexpression of EtRPL27 and EtRPP0 in sensitive strains enhances the resistance of E. tenella to maduramycin, and can be used as molecular markers for Eimeria's resistance to maduramycin. Detecting the expression of EtRPL27 and EtRPL0 can determine E. tenella's resistance to maduramycin. The primers used are EtRPL27 (RPL27-F: 5'-ACAGGGACCTTTTGTTCTCG-3', RPL27-R: 5'-TCGTTTCCAGACCTTTGTCG-3');

[0123] EtRPP0(RPP0-F:5'-GGCGACAGTGCTGATGGGAAAG-3';

[0124] RPP0-R:5'-CAGAAGATGAAGCCGACGTTGAGG-3'). In vitro antibody inhibition tests showed that anti-EtRPL27 and EtRPL0 antibodies have the activity of resisting E. tenella from invading host cells. After overexpression of EtRPL27 and EtRPP0 in sensitive strains, the invasion and pathogenicity of the strains to host cells were enhanced. Therefore, EtRPL27 and EtRPP0 are not only involved in the normal function of E. tenella ribosomes - protein synthesis, but also in other important regulatory functions such as the invasion process of the worm to host cells and the production of maduramycin resistance by the worm. They can be used as molecular markers for the establishment of rapid detection methods for chicken coccidia resistance, targets for the development of drugs for the prevention and treatment of chicken coccidiosis, and target molecules for new vaccines.

[0125] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of a ribosomal protein L27 of Eimeria tenella and / or an acidic ribosomal phosphorylated protein P0 of the large subunit of 60S of Eimeria tenella in the preparation of a preparation for improving the pathogenicity of Eimeria tenella and / or a preparation for resisting the invasion of Eimeria tenella.

2. A method for increasing the pathogenicity of Eimeria, characterized in that: The method comprises over-expressing ribosomal protein L27 and / or 60S large subunit acidic ribosomal phosphorylation protein P0 in E. tenella.

3. The method of claim 2, wherein increasing the pathogenicity of Eimeria refers to increasing the in vitro invasion rate of cells and enhancing the pathogenicity to chickens.

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