CN110129343B - Eimeria tenella superoxide dismutase and application thereof - Google Patents
Eimeria tenella superoxide dismutase and application thereof Download PDFInfo
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- CN110129343B CN110129343B CN201910428573.7A CN201910428573A CN110129343B CN 110129343 B CN110129343 B CN 110129343B CN 201910428573 A CN201910428573 A CN 201910428573A CN 110129343 B CN110129343 B CN 110129343B
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Abstract
The invention relates to Eimeria tenella superoxide dismutase and application thereof, and discloses a molecular marker EtSOD for assisting in identifying drug resistance of Eimeria tenella. The molecular marker EtSOD is a cDNA fragment obtained by taking the genomic cDNA of the Eimeria tenella to be detected as a template and carrying out PCR amplification by adopting a primer F and a primer R. Experiments prove that the molecular marker EtSOD can assist in identifying the drug resistance of the Eimeria tenella to be detected, has the characteristics of rapidness, low cost, less limitation and the like, and can greatly improve the screening efficiency. The invention has great application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular marker EtSOD for assisting in identifying drug resistance of Eimeria tenella to be detected.
Background
Coccidiosis in chickens is a parasitic disease caused by the intestinal tract of eimeria coccidiosis chickens, and causes serious economic loss to the poultry industry all over the world. The medicines are mainly used for preventing and treating coccidiosis and are mainly divided into two types, one is an ionophore medicine influencing the coccidian cell membrane permeability, such as maduramicin, monensin and the like; another class is chemically synthesized drugs that affect coccidial metabolic pathways, such as diclazuril. With the use of a large amount of medicines, the coccidia is extremely easy to generate the drug resistance, and the chicken coccidia generates the drug resistance to almost all the used anticoccidial medicines at present. The generation of drug resistance causes the difficulty of on-site administration and causes great economic loss to the breeding industry all over the world.
At present, the detection of the coccidian drug resistance in the field mainly depends on animal drug susceptibility tests, which are time-consuming, labor-consuming and long in period, so that the research of the rapid detection method of the coccidian drug resistance is particularly important for guiding the field medication. However, as the mechanism of coccidian drug resistance generation is not clear so far, a representative target molecule related to coccidian drug resistance is not found, a rapid detection method is not established, and guidance cannot be provided for field medication. Therefore, the screening and the utilization of the molecular markers related to the drug resistance of the Eimeria tenella have important significance.
Disclosure of Invention
In the early stage of the laboratory, transcriptome sequencing is carried out on sensitive strains and drug-resistant strains of the Eimeria tenella (Eimeria tenella) and drug-resistant strains (a diclazuril-resistant strain and a maduramicin-resistant strain), differential expression genes between the drug-resistant strains and the sensitive strains are screened out, wherein Eimeria tenella superoxide dismutase (EtSOD) is included, and the expression level of EtSOD in the two drug-resistant strains is obviously higher than that of the sensitive strains. Superoxide dismutase is an enzyme which can catalyze superoxide to be converted into oxygen and hydrogen peroxide through a disproportionation reaction, and can remove various active oxygen generated by cell metabolism, so that the aim of protecting cells from being poisoned by oxygen free radicals is fulfilled. It has been found that Toxoplasma and Plasmodium SOD may be involved in the growth and development of insect in host cells, and that superoxide dismutase has been shown to have potential as a drug target for eukaryotic pathogens.
The invention firstly protects a specific primer pair which can be composed of two primers for amplifying specific DNA fragments; the specific DNA fragment has a target sequence of a primer pair consisting of a primer F and a primer R in EtSOD;
the primer F can be a 1) or a 2) as follows:
a1 A single-stranded DNA molecule represented by SEQ ID NO.4 or SEQ ID NO.6 of the sequence Listing;
a2 A DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO.4 or SEQ ID NO.6 and having the same function as SEQ ID NO.4 or SEQ ID NO. 6;
the primer R can be a 3) or a 4) as follows:
a3 A single-stranded DNA molecule represented by SEQ ID NO.5 or SEQ ID NO.7 of the sequence Listing;
a4 DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO.5 or SEQ ID NO.7 and having the same function as SEQ ID NO.5 or SEQ ID NO. 7.
The specific primer pair can specifically consist of the primer F and the primer R.
The invention also protects the application of a specific primer pair. The application of the specific primer pair can be any one of the following b 1) -b 3):
b1 Assisted identification of drug resistance of the Eimeria tenella to be tested;
b2 Auxiliary screening of Eimeria tenella species having or suspected of having drug resistance;
b3 ) to assist in screening for species of Eimeria tenella that do not or are suspected to be non-resistant.
The invention also provides a kit containing any one of the specific primer pairs.
The application of the kit also belongs to the protection scope of the invention. The application of the kit can be any one of the following c 1) -c 3):
c1 ) assisting in identifying the drug resistance of the Eimeria tenella to be detected;
c2 Auxiliary screening of Eimeria tenella species having or suspected of having drug resistance;
c3 Assisted selection of Eimeria tenella species that do not or are suspected to be non-resistant.
The invention also protects a DNA fragment obtained by using cDNA reverse transcribed from mRNA of Eimeria tenella to be detected as a template and adopting any one of the specific primer pairs for amplification.
The DNA fragment is the molecular marker EtSOD to be protected.
The nucleotide sequence of the DNA fragment can be specifically shown as SEQ ID NO.1 or SEQ ID NO.2 in a sequence table.
The amino acid sequence coded by the DNA fragment can be specifically shown as SEQ ID NO.3 in a sequence table.
The application of the DNA fragment also belongs to the protection scope of the invention. The application of the DNA fragment can be any one of the following d 1) -d 8):
d1 Assisted identification of drug resistance of the Eimeria tenella to be tested;
d2 Auxiliary screening of Eimeria tenella species having or suspected of having drug resistance;
d3 ) to assist in screening for species of Eimeria tenella that do not or are suspected to be non-resistant.
d4 Preparing a product for assisting in identifying the drug resistance of the Eimeria tenella to be detected;
d5 Preparing a product for assisting in screening the Eimeria tenella species having or suspected of having drug resistance;
d6 Preparing a product for assisting in screening of eimeria tenella species that do not have or are suspected to have drug resistance;
d7 As a molecular marker.
The invention also discloses a method for assisting in identifying the drug resistance of the Eimeria tenella to be detected, which comprises the following steps:
(1) Taking mRNA reverse transcription products of the Eimeria tenella to be detected as templates, and carrying out PCR amplification by adopting the specific primer pair to obtain PCR amplification products;
(2) After the step (1) is completed, linking the PCR product to a prokaryotic expression vector, and expressing the recombinant protein in a prokaryotic expression system to prepare the polyclonal antibody or the monoclonal antibody. The prepared antibody is adopted to carry out western-blot detection on the Eimeria tenella protein.
(3) After the step (2) is completed, the following judgment is carried out: if the EtSOD protein in the Western-blot detection is higher than the sensitive strain in the Eimeria tenella (field isolate) to be detected, the Eimeria tenella to be detected has or is suspected to have drug resistance, and if the EtSOD protein in the Western-blot detection is lower than or equal to the sensitive strain in the Eimeria tenella (field isolate) to be detected, the Eimeria tenella to be detected does not have or is suspected to have the drug resistance.
The invention also discloses a method for assisting in identifying the drug resistance of the Eimeria tenella to be detected, which comprises the following steps:
(1) Taking cDNA of the Eimeria tenella to be detected as a template
(2) Carrying out quantitative PCR by adopting the specific primer pair;
(3) After the step (2) is completed, the following judgment is carried out: if the transcription content of the mRNA in the quantitative PCR amplification product is higher than that of the sensitive strain in the Eimeria tenella (field isolate) to be detected, the Eimeria tenella to be detected is judged to have drug resistance or is suspected to have drug resistance, and if the transcription content of the mRNA in the quantitative PCR amplification product is lower than or equal to that of the sensitive strain in the Eimeria tenella (field isolate) to be detected, the Eimeria tenella to be detected does not have the drug resistance or is suspected to have no drug resistance.
Experiments prove that the molecular marker EtSOD provided by the invention has higher specificity, can assist in identifying the drug resistance of the Eimeria tenella to be detected, has the characteristics of rapidness, low cost, less limitation and the like, and can greatly improve the screening efficiency. The invention has great application value.
Drawings
FIG. 1: bioinformatics analysis of the nucleotide sequence of the EtSOD gene cDNA and its amino acid sequence, wherein, lower case: an N-glycosylation site; lower case bold font: manganese/iron superoxide dismutase; wave line: casein kinase II phosphorylation site; underlining: an N-myristoylation site; and (3) inclining the font: a protein kinase C phosphorylation site; the method comprises the following steps: an iron/manganese superoxide dismutase alpha-hairpin domain; shading: a tyrosine kinase phosphorylation site; the method comprises the following steps: iron/manganese superoxide dismutase C-terminal domain; asterisks: and a terminator.
FIG. 2 purification of recombinant protein rEtSOD
Figure 3 distribution localization of EtSOD, where a: sporozoites resuspended in PBS, B: sporozoites resuspended in PBS, C: invasion of sporozoites into DF-1 cells 2h, D: invasion of the DF-1 cells by sporozoites 24h, E: invasion of sporozoites into DF-1 cells 48h, F: invasion of DF-1 cells by sporozoites 60h G: invasion of sporozoites into DF-1 cells 72h, H: DF-1 cells invaded by sporozoites for 84h
Figure 4 immunogenicity assay of rtesod, where M: protein Marker,1: primary antibody-negative sera, 2: the primary antibody is a His tag monoclonal antibody, 3: the primary antibody is rabbit anti-sporozoite polyclonal antibody
FIG. 5 transcriptional levels of EtSOD gene at different developmental stages of polypide
Fig. 6 transcriptional and translational levels of EtSOD gene, where a: etSOD gene transcript levels; b: etSOD Gene translation level
Fig. 7 transcriptional and translational levels of EtSOD gene, where a: the transcriptional level of the EtSOD gene; b: etSOD gene translation level. And (2) DS: eimeria tenella sensitive strain 1: under the action of 0.2ppm diclazuril, 2:0.5ppm diclazuril, 3:0.8ppm diclazuril.
Fig. 8 transcriptional and translational levels of EtSOD gene, where a: etSOD gene transcript levels; b: etSOD gene translation level. And (2) DS: eimeria tenella sensitive strain 1:3ppm Maduramycin 2: under the action of 5ppm maduramicin
FIG. 9 EtSOD enzyme activity assay.
Examples
The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 cloning and bioinformatic analysis of EtSOD Gene
Insect strain
The bodies of Eimeria tenella Shanghai strain (resource number: CAAS 21111601), the drug-resistant strains of Declazuril (resource number: CAAS 2111160821) and the drug-resistant strains of maduramicin (resource number: CAAS 2111160921), and the different drug concentrations (0.2 ppm, 0.5ppm and 0.8ppm of Declazuril-resistant strains; 3ppm and 5ppm of maduramicin-resistant strains) were isolated, induced and stored by the Protozoa Innovation team at the Shanghai veterinary institute of academy of sciences of China agricultural sciences. Purified non-sporulated oocysts (UO), sporulated Oocysts (SO), sporozoites (SZ) and second generation merozoites (SM) were obtained by reference following mass propagation of 2 weeks old coccidiless chicks.
RNA extraction and cDNA preparation
Total RNA from worms was extracted using TRIzol reagent according to the instructions. Total RNA concentration was determined using a spectrophotometer at 260 nm. RNA quality was assessed by electrophoresis on a 1% agarose gel. Complementary DNA (cDNA) was synthesized from total unsporolated oocyst RNA using Super Script III RT reagent.
Amplification of
Upstream primers 5' -GC with restriction sites BamHI and XhoI (underlined), respectively, were usedGGATCCATGGCG CTCCGCAGCCGCCCTTCGCCTCA-3 '(SEQ ID NO. 4) and downstream primer 5' -GCCTCGA GTCAGTAGGTCTCGAGGAGGTTGAGGAACCTGTTC-3' (SEQ ID NO. 5) to amplify the ORF sequence of the EtSOD gene. cD Using Nosporulated oocystsNA was used as a template for PCR amplification. Total 25, μ L, procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and continuous extension at 72 ℃ for 10min. The PCR product was detected by 1% agarose gel electrophoresis and the target band was recovered using QIAquick gel extraction kit. The recombinant plasmid pGEM-T-EtSOD was constructed by ligating the EtSOD fragment to a pGEM-T-easy vector using T4DNA ligase. The recombinant plasmid is sent to a living organism (Shanghai, china) for sequencing and identification. And (3) performing PCR amplification by taking the first strand of the cDNA of the sporulated oocyst of the sensitive strain of the Eimeria tenella as a template. The resulting band size was identical to that expected, approximately 651bp, and was named EtSOD. The nucleotide sequence of the obtained EtSOD is shown in SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 3. Meanwhile, the genome of the Shanghai strain of Eimeria tenella (resource number: CAAS 21111601) is taken as a template to carry out PCR amplification, a specific PCR system and steps are as described above, the full-length nucleotide sequence of the obtained EtSOD is shown as SEQ ID NO.2, and the lowercase letters are intron sequences therein. And after connecting, transforming and sequencing the target fragment and a pGEM-T-easy vector, screening positive clones, and performing sequence determination and bioinformatics analysis on the positive clones.
Bioinformatics analysis
The molecular weight and theoretical isoelectric point of EtSOD were predicted using the ProtParam tool on ExPASY (http:// web. ExPASy. Org/ProtParam /). The presence or absence of signal peptide in the encoded protein was predicted using the calculation tool SignalP (http:// www.cbs.dtu.dk/services/SignalP /) and TMHMM (http:// www.cbs.dtu.dk/services/TMHMM-2.0 /) for transmembrane structure in the encoded protein. Protein motifs were analyzed using a Motif Scan (http:// hits. Isb-sib. Ch/cgi-bin/Motif _ Scan). And (3) analyzing and displaying by using bioinformatics software: the gene sequence is 651bp in length, 216 amino acids are coded, the predicted molecular mass is 24.9kDa, and the isoelectric point is 7.15; the analysis of the signal peptide and the transmembrane structure shows that the protein expressed by the gene sequence has no signal peptide and no transmembrane structure; the structural functional domain of the protein was found that the protein encoded by the gene contained 1 phosphorylation site of casein kinase II (site positions: 144-147), 2N-myristoylation sites (site positions: 74-79 and 116-121), 4 phosphorylation sites of protein kinase C (site positions: 11-13, 31-33, 107-109 and 146-148), 2N-glycosylation sites (site positions: 85-88 and 187-190), 1 phosphorylation site of tyrosine kinase (site positions: 33-39), 1 iron/manganese superoxide dismutase, C-terminal domain (site positions: 101-208), 1 iron/manganese superoxide dismutase, an alpha-hairpin domain (site positions: 13-94) and 1 manganese and iron superoxide dismutase (site positions: 174-181) (FIG. 1). EtSOD proteins belong to the superoxide dismutase family.
Example 2 expression of the recombinant protein EtSOD
pGEM-T-EtSOD and pET-28a vectors were subjected to double digestion with BamHI/XhoI, recovered in agarose gel, ligated, and transformed into E.coli BL21 (DE 3) with the recombinant pET-28a-EtSOD plasmid. Carrying out amplification culture on the colony with the correct sequencing at 37 ℃, adding 1.0mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) when the OD value is between 0.6 and 1.0, placing the colony in a constant-temperature shaking table at 16 ℃ for shake culture for 24 hours, and keeping the rotation speed of the shaking table at 180r/min. And ultrasonically cracking the thalli in ice bath for 2s by ultrasonic, stopping for 2s, and continuing for 20min. And purifying recombinant protein rEtSOD containing His labels by adopting a His-Bind Resin affinity column chromatography method. SDS-PAGE electrophoretic detection shows that relatively pure fusion protein is obtained, as shown in FIG. 2. Protein concentration was determined using BCA protein assay kit. The protein was stored at-20 ℃ for subsequent analysis.
Example 3 localization of protein EtSOD distribution on insect bodies
Preparation of rEtSOD antiserum
New Zealand white rabbits, 2 months old, were immunized subcutaneously after emulsification of 200. Mu.g of purified rEtSOD with an equal volume of Freund's complete adjuvant. Two weeks later, a booster was performed, and the booster was emulsified with Freund's incomplete adjuvant. Immunizations were performed every 7 days for a total of 5 immunizations. 7 days after the last immunization, blood was collected and polyclonal antibody serum was isolated. Negative sera were isolated from rabbits following blood collection from the ear vein prior to immunization.
Distribution of EtSOD in different developmental stages of Eimeria tenella
According to 2X 10 per hole 5 The DF-1 cells are inoculated in a six-well plate with a cell slide, and 2 is addedmL of complete medium (containing 10% fetal bovine serum, 100 units/mL of double antibody) was cultured in a cell culture chamber at 37 ℃ for 24 hours. Fresh E.tenella sporozoites (6X 10 per well) 5 Parasites) DF-1 cells were infected after 2 hours of incubation at 41 ℃ in complete medium and cell crawls were collected at 24, 48, 60, 72 and 84 post infection. All samples were fixed with 4% paraformaldehyde for 20min at room temperature, permeabilized with 1% Triton X-100 for 15min, and then blocked with 2% bovine serum albumin in a refrigerator at 4 ℃ overnight. The samples were incubated with rabbit anti-rEtSOD polyclonal antibody at a ratio of 1: 100 for 2 hours at 37 deg.C, followed by incubation with isothiocyanate (FITC) fluorescein labeled goat anti-rabbit IgG (1: 500 dilution) for 1h at 37 deg.C. Nuclei were stained with 15. Mu.g/ml 4, 6-diamidino-2-phenylindole (DAPI) for 30min. After each incubation, all samples were washed 4 times with PBS. Sporozoites and second generation merozoites were evenly spread on a cell slide. Then, after fixation with 4% paraformaldehyde, blocking with 1% Triton X-100 and 2% bovine serum albumin, the sample was incubated with rabbit anti-rEtSOD polyclonal antibody as primary antibody and FITC as secondary antibody, and nuclei were labeled with DAPI. After that, 60 μ L of the anti-fluorescence quencher was dropped on the slide glass (cell slide was reversely buckled on the slide glass. Photograph was observed by confocal laser fluorescence microscope. The results (fig. 3) showed that the expression amount of EtSOD protein was small on the sporozoites and the second generation merozoites, and the green fluorescence intensity of both the sporozoites and the second generation merozoites was weak (fig. 3A and B). After infecting DF-1 cells with the sporozoites for 2H and 24H, etSOD was mainly localized at the top of the polypide, and the green fluorescence intensity was also increased at this stage (fig. 3C and D). At 48H, we found that the expression amount was decreased at the trophozoite stage, the fluorescence intensity was decreased (fig. 3E). As the intracellular polypide developed, etSOD was localized on the whole surface of the immature merozoites and mature merozoites, and the expression amount was increased (fig. 3f, g and H).
Example 4 immunogenicity analysis of the recombinant protein rEtSOD
Fluorescent quantitative PCR
To test the transcriptional level of EtSOD in different developmental stages of Eimeria tenella (non-sporulated oocysts, sporozoites and second generation merozoites), and to compare sensitive strainsDifferences in transcription levels between sporulated oocysts from drug-resistant strains of different drug concentrations (diclazuril-resistant and maduramicin-resistant strains). After extraction of worm RNA and removal of the genome, the first cDNA strand was synthesized using SuperScript III reverse transcriptase and random primers. The transcript level of EtSOD was detected using SYBR GreenI using the Worm cDNA as a template, using the upstream primer 5 'AGATTCTCTCTGCCGCCGCTCCCCCT-3' (SEQ ID NO. 6) and the downstream primer 5 'GCGTTCAAGTTGTCCACATAAGCT-3' (SEQ ID NO. 7). Amplification was performed using 18S ribosomal RNA of Eimeria tenella as a reference, and using 5 '-upstream primer-TGTAGTGGAGTCTTGGTGATTC-3' and 5 '-downstream primer-CCTGCTTCCTTAGATGA-3'. 3 replicates per sample and the experiment was repeated three times. Use 2 -ΔΔCt The method calculates the relative expression level of EtSOD.
Western blot
For detecting immunogenicity analysis of recombinant proteins, rEtSOD recombinant proteins were subjected to SDS-PAGE, transferred to PVDF membrane, 5% skimmed milk powder overnight at 4 deg.C, washed 3 times with PBS for 5min, incubated with anti-sporozoite serum (1: 100 dilution), his mab (1: 2000 dilution) and rabbit IgG as primary antibody at 37 deg.C for 2h, washed 3 times with PBS, and then incubated with rEtSOD recombinant proteins
680RD donkey anti-mouse IgG or
680CW goat anti-rabbit IgG (diluted 1: 10 000) was incubated in the dark for 1h, washed with PBST for 5 times, and scanned with a two-color infrared laser imaging system. The results of scanning in the two-color infrared laser imaging system (fig. 4) showed that bands appeared at the desired sites in the His monoclonal antibody, anti-sporozoite holoprotein polyclonal antibody as the primary incubated membrane. While the rabbit negative serum did not show bands at the target sites as the primary antibody incubated membrane. The recombinant protein rEtSOD has good immunogenicity.
The expression of EtSOD protein in sporulated oocysts of sensitive strains and drug-resistant strains (diclazuril-resistant strain and maduramicin-resistant strain) with different drug concentrations was analyzed in the same manner as described above. Taking a proper amount of sporulated oocysts from each group, suspending the sporulated oocysts in 0.5mL of cell lysate, adding a protease inhibitor to make the final concentration of the protease inhibitor 1%, adding glass beads, and placing the mixture on a vortex oscillator to shake for 60min, wherein each shaking time is 8min, and the mixture is subjected to ice bath for 2min. Protein extraction was checked by SDS-PAGE and BCA was used to determine protein concentration in each worm, and Western blot was performed on equal amounts of protein from each group, where the primary antibody was rEtSOD anti-rabbit serum, while the α -tubulin monoclonal antibody was used as an internal control. The results show that the gene mRNA is transcribed at the highest level in the second generation merozoite (Mrz), and the non-sporulated oocysts (UO) are counted next time. Sporulated Oocysts (SO) and sporozoites (Spz) were lowest (fig. 5). Furthermore, using real-time fluorescent quantitative PCR detection, the transcriptional level of the EtSOD gene was found to be significantly upregulated in the diclazuril-resistant strain (DZR) and the maduramicin-resistant strain (MRR) compared to the eimeria tenella-sensitive strain. Differential analysis of transcription and translation levels in sensitive and resistant strains confirmed that the housekeeping gene alpha-Tubulin was used as an internal reference, and Western blotting detection was used to find that the translation level of EtSOD protein in two resistant strains is significantly up-regulated compared with that of the sensitive strain of Eimeria tenella (see FIG. 6). Moreover, the analysis of the transcriptional and translational levels of the EtSOD gene under the action of diclazuril with different concentrations proves that the transcriptional level of the EtSOD gene is higher along with the increase of the drug concentration. The housekeeping gene alpha-Tubulin is used as an internal reference, and is found by Western blotting detection: the level of EtSOD protein translation also increased with increasing drug concentration. As shown in fig. 7. Analysis of the transcription and translation levels of the EtSOD gene under the action of maduramicin with different concentrations proves that Western blotting detection shows that the translation level of the EtSOD protein is positively correlated with the drug concentration. As shown in fig. 8.
Enzymatic activity assay
The enzymatic activity difference of EtSOD among sporulated oocysts of a sensitive strain, a diclazuril-resistant strain and a maduramicin-resistant strain was detected by using a superoxide dismutase (SOD) kit developed by the Japan institute of Dorem chemistry. The principle is to utilize highly water-soluble tetrazolium salt developed by the same institute of chemistry
(2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid phenyl)) -2-hydro-tetrazolium salt, disodium salt) with superoxide anion (O2-) to form a water-soluble dye.
The reduction ratio by superoxide anion is linearly related to the xanthine oxidase activity, and is inhibited by SOD (SOD reaction occurs preferentially,
occurring after the reaction) to exclude its own effect on the experiment. The specific operation steps refer to the use specification. The superoxide dismutase detection kit is used for carrying out three independent experiments on the basis of a preliminary experiment, and the experimental result shows that the activity of EtSOD enzyme in a diclazuril resistant strain (DZR) and a maduramicin resistant strain (MRR) is obviously improved compared with that of a sensitive strain. As shown in fig. 9.
Claims (3)
1. The application of the molecular marker for assisting in identifying the diclazuril resistance and the maduramicin resistance of the Eimeria tenella to be detected can be any one of the following 1) -3):
1) Assisting in identifying the drug resistance of the Eimeria tenella to be detected and the drug resistance of maduramicin;
2) Auxiliary screening of Eimeria tenella species having or suspected of having diclazuril resistance and maduramicin resistance;
3) Auxiliary screening of Eimeria tenella species which do not have or are suspected to have diclazuril resistance and maduramicin resistance;
the molecular marker is an Eimeria tenella superoxide dismutase gene (EtSOD), and the nucleotide sequence of the EtSOD is shown as SEQ ID NO.1 or SEQ ID NO. 2; the amino acid sequence is shown as SEQ ID NO. 3;
the application specifically comprises the steps of utilizing a specific primer pair to amplify diclazuril resistance and maduramicin resistance molecular marker target sequences of the Eimeria tenella;
the primer F is a single-stranded DNA molecule shown in SEQ ID NO.4 or SEQ ID NO.6 of the sequence table;
the primer R is a single-stranded DNA molecule shown by SEQ ID NO.5 or SEQ ID NO.7 of a sequence table.
2. A method for assisting in identifying the drug resistance of Eimeria tenella and the drug resistance of maduramicin comprises the following steps:
(1) Carrying out PCR amplification by using the mRNA reverse transcription product of the Eimeria tenella to be detected as a template and adopting the specific primer pair in the claim 1 to obtain a PCR amplification product;
(2) After the step (1) is completed, linking the PCR product to a prokaryotic expression vector, and expressing recombinant protein in a prokaryotic expression system to prepare a polyclonal antibody or a monoclonal antibody; carrying out western-blot detection on the Eimeria tenella protein by adopting the prepared antibody;
(3) After the step (2) is completed, the following judgment is carried out: if the separated plant of the EtSOD protein in the field of the Eimeria tenella to be detected in the Western-blot detection is higher than the sensitive plant, the Eimeria tenella to be detected is judged to have or be suspected to have the drug resistance of the diclazuril and the drug resistance of the maduramicin, if the separated plant of the EtSOD protein in the field of the Eimeria tenella to be detected in the Western-blot detection is lower than or equal to the sensitive plant, the Eimeria tenella to be detected does not have or is suspected to have the drug resistance of the diclazuril and the drug resistance of the maduramicin.
3. A method for assisting in identifying the drug resistance of Eimeria tenella and the drug resistance of maduramicin comprises the following steps:
(1) Taking the first cDNA chain of the Eimeria tenella to be detected as a template
(2) Carrying out quantitative PCR by using the specific primer pair in the claim 1;
(3) After the step (2) is completed, the following judgment is carried out: if the transcription content of mRNA in the quantitative PCR amplification product is higher than that of a sensitive strain in the field of the to-be-detected Eimeria tenella, the to-be-detected Eimeria tenella is judged to have or is suspected to have the drug resistance of diclazuril and the drug resistance of maduramicin, and if the transcription content of mRNA in the quantitative PCR amplification product is lower than or equal to that of the sensitive strain in the field of the to-be-detected Eimeria tenella, the to-be-detected Eimeria tenella does not have or is suspected to have the drug resistance of diclazuril and the drug resistance of maduramicin.
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| 柔嫩艾美耳球虫耐药相关基因特性初步研究及耐药株与敏感株全基因组重测序分析;陈婷;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20190115;全文 * |
| 柔嫩艾美耳球虫耐药相关基因的分析鉴定;刘桂玲;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20190815;全文 * |
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