CN111965353B

CN111965353B - Application of psoroptes ovis cysteine protease inhibitor and ELISA kit

Info

Publication number: CN111965353B
Application number: CN202010834641.2A
Authority: CN
Inventors: 古小彬; 王策; 谢跃; 杨光友; 何冉; 彭雪蓉
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-08-19
Anticipated expiration: 2040-08-18
Also published as: CN111965353A

Abstract

The invention relates to the technical field of biology, and discloses a series of related applications of a psoroptes ovis cysteine protease inhibitor as a psoroptes disease and/or sarcoptidosis diagnostic antigen, and the like, wherein related experimental results show that the psoroptes ovis cysteine protease inhibitor can be identified by positive rabbit serum of psoroptes ovis and sarcoptidosis, and the result shows that the psoroptes ovis cysteine protease inhibitor has reactogenicity; meanwhile, the sheep psoroptes cystatin has higher sensitivity and specificity in an indirect ELISA method (rPCys-iELISA) established by the recombinant cystatin, and various results prove that the sheep psoroptes cystatin can be used as a diagnostic antigen of psoroptes and/or sarcoptidosis and applied to a related detection kit.

Description

Application of psoroptes ovis cysteine protease inhibitor and ELISA kit

Technical Field

The invention relates to the technical field of biology, in particular to application of a psoroptes ovis cysteine protease inhibitor and an ELISA kit.

Background

As one of the world's largest rabbit breeding producing countries, according to statistics of rabbit industry system data in 2015, the total yield and export of rabbit skin, rabbit hair and rabbit meat in China stably live in the first world for many years. The psoriasis is one of the common ectoparasite diseases in rabbit breeding, and the reported infection rate of domestic rabbits is up to 70%. The disease is caused by itch mite ovis of sheep parasitizing on the skin surface of the external auditory canal of a rabbit, shows itching, ear scratching and escharosis of a paper sample in the external auditory canal as main clinical symptoms, causes the weight reduction of the rabbit, the reduction of feed conversion rate and the reduction of immune function, leads severe patients to die, and causes huge economic loss to rabbit industry. Therefore, the animal suffering from the psoroptes can be used for timely and accurately diagnosing the psoroptes to prevent the propagation of the psoroptes in rabbit groups, and the economic loss and the animal welfare of the rabbit raising industry can be improved.

Early scrapie ovis infection in rabbits had no obvious clinical symptoms, and this subclinical case lasting several weeks was the source of infection for the colony. Currently, the scab microscopic examination by scraping is still the gold standard for definite diagnosis of the psoriasis, the diagnosis of the psoriasis in the rabbit industry still depends on observing clinical symptoms and microscopic examination of mites in scabs in a skin damage area, but the sensitivity of the body microscopic examination of the scab can be as low as 18 percent, and the traditional diagnosis method is easy to miss the cases of subclinical infection and slight infection, so that serious disease prevalence is caused; moreover, the diagnosis of etiology is difficult to be truly clinically applied due to its low efficiency and workload, susceptibility to missed detection of animals infected with subclinical symptoms or low mite population, and time-consuming. Therefore, the animal infected with the psoroptes ovis can be timely and accurately diagnosed, and the method is favorable for earlier and more effective treatment, prevention and control of the psoroptes ovis.

Serum antibodies can be raised in animals infected with itch mite and such antibodies can appear before the animals have a significant clinical manifestation. Serological diagnosis is rapidly developing in the diagnosis of parasites today due to its advantages of high accuracy and low workload. Therefore, the ELISA method can make up for the defects of the traditional etiology diagnosis method and becomes a diagnosis method for the psoriatic mite disease subclinical case. However, no diagnostic kit for psoriatic mite of rabbits is available at present. Therefore, the present invention aims to develop an effective serodiagnostic method for psoriatic mite.

Disclosure of Invention

In view of the above, the present invention aims to provide a psoroptes ovis cysteine protease inhibitor as a diagnostic antigen for psoroptes ovis and/or sarcoptidosis and an application thereof in preparing the diagnostic antigen for psoroptes ovis and/or sarcoptidosis, such that the psoroptes ovis cysteine protease inhibitor has high specificity and sensitivity;

another object of the present invention is to provide an application of the cysteine protease inhibitor of psoroptes ovis in the preparation of a kit for detecting psoroptes and/or sarcoptidosis, such that the ELISA method established by the cysteine protease inhibitor of psoroptes ovis shows high specificity and sensitivity, and can be used for ELISA detection;

another object of the present invention is to provide an ELISA kit for diagnosing psoriasis and/or sarcoptidosis, which has high specificity and sensitivity in detection;

currently, the search for cysteine protease inhibitors as candidate diagnostic antigens is more developed in the field of parasitic diseases, such as: fasciola hepatica, toxoplasma, cysticercus, etc. However, no relevant studies of cysteine protease inhibitors in psoriatic or sarcoptidosis have been found.

Herein, the psoroptes ovis cysteine protease inhibitor may be non-natural, e.g. synthetic or expressed from an artificial vector (commonly referred to in the art as the recombinant protein rPCys). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance.

The invention clones and expresses the cysteine protease inhibitor gene (PCys) of the psoroptes ovis and carries out molecular identification on the gene by bioinformatics analysis. The immunogenicity of recombinant protein cysteine protease inhibitors (rPCys) was studied by immunoblotting, and an indirect ELISA method was established to evaluate its potential for serological diagnosis of psoriatic rabbit. The result shows that the full length of ORF frame of PCys gene is 396bp, the recombinant fusion protein cystatin obtained by prokaryotic expression is about 33.3kDa, and can be identified by the serum of positive rabbit suffering from itch mite disease, which shows that the recombinant fusion protein has reactogenicity; the optimal coating concentration of the indirect ELISA method (rPCys-iELISA) established by the recombinant cystatin is 8.33 mu g/mL, the serum dilution is 1:200, the secondary antibody dilution is 1:3000, the critical value is 0.358, and the sensitivity and the specificity are both 93.75% (45/48).

Based on the test result, the invention provides the application of the cysteine protease inhibitor of the psoroptes ovis as a diagnostic antigen of the psoroptes ovis and in the preparation of the diagnostic antigen of the psoroptes ovis; and provides the application of the psoroptes ovis cysteine protease inhibitor in the preparation of a kit for detecting psoroptes ovis; in a particular embodiment of the invention, the psoriatic mite is described by way of example for rabbit psoriatic mite.

Preferably, the kit is an ELISA kit; in a specific embodiment, the ELISA kit is a kit based on an ELISA indirect method.

In addition, according to the application, the invention also provides an ELISA kit for diagnosing the psoroptes, which comprises a solid phase carrier coated with the cysteine protease inhibitor of the psoroptes ovis. In a specific embodiment of the invention, the solid phase carrier can be selected from a 96-well culture plate or a similar solid phase carrier, the optimal coating concentration of the psoroptes ovis cysteine protease inhibitor is 8.33 mug/mL, and the carrier can be coated by a coating solution which is 0.39g Na ₂ CO ₃ ，35mMNaHCO ₃ 0.2M NaCl, adjusted to pH 9.6.

After the core components of the kit are determined, the ELISA kit further comprises one or more than two of enzyme-labeled secondary antibody, washing liquid, developing liquid, confining liquid, diluent and stop solution.

The enzyme-labeled secondary antibody is preferably goat anti-rabbit IgG labeled with HRP, and in the specific embodiment of the invention, the enzyme-labeled secondary antibody is a commercially available product, and the dilution ratio of the enzyme-labeled secondary antibody is 1: 3000A;

the washing solution is preferably PBS-T washing solution, and in the specific embodiment of the invention, the PBS-T washing solution is composed of: 137mM NaCl,2.7mM KCl,10mM Na ₂ HPO ₄ ,2mM KH ₂ PO ₄ 0.1% v/vtween-20, pH 7.4; the color development liquid is preferably TMB color development liquid;

the confining liquid is preferably skimmed milk, in a particular embodiment of the invention, the skimmed milk has a concentration of 5%.

The stop solution is preferably a sulfuric acid solution, and the concentration is preferably 2 mol/L; the preparation method comprises slowly dripping 21.7mL of 98% concentrated sulfuric acid into 178mL of deionized water, cooling to room temperature, and storing at 4 ℃;

the diluent is preferably PBS; the preparation method comprises 8g of NaCl, 0.2g of KCl and 1.42g of Na ₂ HPO ₄ ，0.27gKH ₂ PO ₄ Dissolving in 800mL deionized water, dissolving to 1L, sterilizing, and storing at room temperature.

According to the technical scheme, the invention provides the relative application of the psoroptes ovis cysteine protease inhibitor as a psoroptes diagnostic antigen, and the relative experiment results show that the psoroptes ovis cysteine protease inhibitor can be identified by the serum of positive rabbit suffering from the psoroptes, which shows that the psoroptes ovis cysteine protease inhibitor has reactogenicity; meanwhile, the antipyrotic sheep cystatin has higher sensitivity and specificity in an indirect ELISA method (rPCys-iELISA) established by the recombinant cystatin, and various results prove that the antipyrotic sheep cystatin can be used as a diagnostic antigen of the psoroptes, particularly the psoroptes rabbit, and can be applied to a related detection kit.

Drawings

FIG. 1 shows PCR amplification of PCys; lane M represents the relative molecular mass standard of DNA, lane 1 represents the PCys amplification product, and 2 represents the negative control;

FIG. 2 shows plasmid double restriction enzyme identification; wherein, a: lane M represents the relative molecular mass standard of DNA, and

lanes

1 and 2 represent the recombinant plasmid pMD19-T-PCys double cleavage product; b: lane M represents the relative molecular mass standard of DNA, and

lanes

1 and 2 represent the plasmid pET32a-PCys double cleavage products;

FIG. 3 shows an immunoblot analysis of rPCys; lane M represents the relative quality standard of protein, Lane 1 represents the expression of plasmid pET32a (+) bacterial liquid induced by IPTG and the staining product of Coomassie brilliant blue, Lane 2 represents the expression of recombinant plasmid pET32a (+) -PCys bacterial liquid induced by IPTG and the ultrasonic disruption, the precipitate is taken and dissolved in 2M urea to obtain the staining product of Coomassie brilliant blue, Lane 3 represents the protein of purified rPCys which is subjected to SDS-PAGE electrophoresis and the staining product of Coomassie brilliant blue, and Lane 4 represents the positive incubation of purified rPCys with rabbit serum infected with psoriases and the staining product of DAB;

FIG. 4 shows sensitivity, specificity and cross-reactivity of rPCys indirect ELISA; the ordinate represents the OD of the sample serum ₄₅₀ The values, abscissa representing different kinds of serum samples, thin horizontal line representing the cut-off value (cut-off value) of positive detection, and short horizontal line representing the OD of the serum sample ₄₅₀ Mean, ns means no significant difference, and x means very significant difference.

Detailed Description

The invention discloses application of a psoroptes ovis cysteine protease inhibitor and an ELISA kit, and a person skilled in the art can use the contents to reference the contents and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The applications and kits of the present invention have been described by way of example, and it will be apparent to those skilled in the art that modifications, or appropriate variations and combinations of the applications and kits described herein can be made to implement and use the techniques of the present invention without departing from the spirit, scope, and spirit of the invention.

The coding sequence of the scrapie cysteine protease inhibitor is amplified from cDNA by extracting mite body RNA and carrying out reverse transcription to obtain the cDNA. After T cloning, the amplified product is introduced into an expression vector in a way of enzyme digestion connection, and prokaryotic expression is carried out by using escherichia coli to obtain recombinant rPCys.

In experiments of the concrete embodiment, all experimental animals were treated strictly in accordance with the "animal protection of the people's republic of china" (draft released on 9/18 of 2009). All procedures were carried out strictly in accordance with the "guidelines for animal Care of the animal ethics Committee of the university of Sichuan agriculture" (China, Yaan; approval No.: 2013-028). All methods are performed according to relevant criteria and specifications, including any relevant details.

The psoroptes bodies come from ear crusts of the rabbit suffering from the psoroptes in Sichuan province, and the psoroptes bodies are collected in a 37 ℃ incubator for 0.5 h. Collecting mite bodies according to morphological characteristics of the itch mite in sheep (reference document: Chenshirong, Lianhongjun, Xuxueying, and the like; periodic observation of the development of itch mite in sheep and research on biological characteristics [ J ] Chinese veterinary science, 2000,30(7): 5-7.);

collecting the antipruritic positive serum from a New Zealand rabbit which is subjected to clinical symptom evaluation and ear crusty skin detection to confirm the diagnosis of the antipruritic acariasis; the negative serum is collected from New Zealand rabbits in a rabbit farm without worm eggs, any clinical diseases and the epidemic history of itch mite; other parasitic disease positive sera: the rabbit positive serum of the scabies and the rabbit coccidian positive serum are both provided by the parasite disease research center of Sichuan university of agriculture (the rabbit serum is from a New Zealand rabbit infected with a single parasite pathogen).

Three biological replicates were performed for all experiments of the invention. All data were analyzed for significance (P < 0.05 considered significant and P < 0.01 considered very significant) and plotted using SPSS Statistics17.0 and GraphPad Prism 7.

Unless otherwise specified, the experimental animals, plasmid vectors, strains, etc. used in the present invention can be obtained commercially.

The application of the cysteine protease inhibitor of psoroptes ovis provided by the present invention and an ELISA kit are further described below.

Example 1: gene amplification and protein expression

1. Gene amplification

Ear crusts of the rabbits infected with the itch mites are placed on a clean plate and placed in an incubator at 37 ℃ for 0.5h to collect itch mite bodies. Collecting mite bodies according to the morphological characteristics of the itch mites in sheep; RNA (TaKaRaMiniBEST Universal RNA Extraction kit) is extracted from the collected mite body and then reverse transcribed into cDNA (PrimeScript) ^TM RT reagent kit with DNA Eraser (Perfect Real Time)). Using touchdown PCR amplification method (table 1), the complete sequence of the ORF box of the PCys gene was amplified (primers see table 2) with an amplification system of 10 μ L: 0.5. mu.L of forward primer, 0.5. mu.L of reverse primer, 0.5. mu.L of cDNA template, ddH ₂ O 3.5μL，2×PCRMixture5μL。

TABLE 1 PCR amplification conditions for the PCys Gene

TABLE 2 primers for PCR

After the PCR product is detected by 1% agarose gel electrophoresis and amplified by PCR, the agarose electrophoresis result shows that a visible target band exists in the interval of 200 bp-500 bp, the size of the visible target band accords with the expectation, and the band is single (figure 1). The gel containing the target strip was cut under an ultraviolet lamp and the target strip was recovered according to the instructions of the gel recovery kit from Beijing Tiangen. Connecting and converting the recovered product of the glue and pMD19-T to form a coated plate, selecting a single bacterial colony to perform bacterial liquid PCR identification, sending the bacterial liquid with a positive result to a biological engineering (Shanghai) corporation Limited for sequencing, and reserving the bacterial liquid with a sequencing result completely consistent with an original sequence.

The gene sequences returned by the company are compared through an NCBI database website, and are translated into amino acid sequences by using a self-online software prediction open reading frame predictor (ORF Finder) (http:// www.ncbi.nlm.nih.gov/gorf. html); predicting the transmembrane region by using TMHMM Sever2.0(http:// www.cbs.dtu.dk/services/TMHMM-2.0); the molecular weight and the isoelectric point pI are predicted by using ExPASY Proteomics Server (http:// web. Expasy. org/protparam /); SignalP server (http:// www.cbs.dtu.dk/Services/SignalP /) was used to predict signal peptides; the online software (http:// tools. immuneepitope. org/bcell /) predicts the epitope; SWISS-MODEL (http:// swissminor. expay. org /) was used to predict protein structure.

The PCys gene contains a complete open reading frame (ORF, the sequence is shown as SEQ ID NO. 1) with 396bp bases, and 131 amino acid residues are coded (the sequence is shown as SEQ ID NO. 2). It is predicted that the PCys total molecular mass is about 15.3kDa, the theoretical isoelectric point is 5.35, there is no transmembrane helix region, there is no signal peptide, 56.49% alpha-helix, 12.98% beta-sheet, 5.34% beta-turn and 25.19% random coil. PCys has typical cysteine protease inhibitor conserved functional sites, similar to cysteine protease inhibitors of house dust mites, european mite and scabies.

2. Protein expression

The plasmid with the correct sequencing was extracted, double-digested with the restriction enzymes shown in Table 2 to obtain the target fragment, and subcloned into pET32a (+) expression vector. The constructed expression vector is transfected into an escherichia coli DH5 alpha competent cell for sequencing, a colony with correct sequencing is selected for amplification culture, then a plasmid is extracted, and then the escherichia coli Rosetta (DE3) is transfected. The transfected E.coli Rosetta (DE3) was grown up and induced for 10h at 20 ℃ with 0.5mM IPTG. The expressed recombinant protein was then purified using a Ni + affinity chromatography device and the collected protein was ultrafiltered and identified by SDS-PAGE and immunoblot analysis.

The immunoblotting method: rPCys proteins were separated using 12% SDS-PAGE and transferred onto nitrocellulose filters (NC membranes); sealing with skimmed milk powder for 2 hr; adding primary antibody (positive serum of itch mite disease rabbit) diluted by PBS according to a ratio of 1:100 in advance, and incubating overnight at 4 ℃; washing with TBST for 5min 3 times; adding secondary antibody (HRP-labeled goat anti-rabbit IgG) which is diluted by PBS at a ratio of 1:3000 in advance, and incubating for 1h at 37 ℃ in a dark place; washing with TBST for 5min 3 times; the color was developed with Diaminobenzidine (DAB), and the reaction was stopped with distilled water until a band appeared.

After double digestion of the recombinant plasmid pMD19-T-PCys (figure 2-a) and the plasmid pET32a (+), the recombinant plasmid pET32a-PCys (figure 2-b) is constructed by connecting and transforming, and agarose electrophoresis results after digestion show that the PCys is inserted into pET32a, and the size of the band is single and accords with the expectation.

The plasmid of pET32a-PCys with correct double enzyme digestion identification and correct sequencing is transformed into escherichia coli Rosetta for prokaryotic expression of target protein, the analysis result is shown in figure (figure 3), a protein band with the size of 33.3kDa can be seen after SDS-PAGE electrophoresis and Coomassie blue staining, the protein band conforms to the expected size, and the protein band is mainly expressed in an inclusion body. The PCys recombinant antigen can be recognized by the serum of the rabbit infected by the itch mite, has a single strip and is consistent with the size and the expectation.

Example 2: establishment of rPCys protein indirect ELISA method

1. Method of operation

Indirect ELISA was performed as described by shen et al. The optimal working conditions of protein coating concentration, serum concentration and secondary antibody (goat anti-rabbit IgG) concentration are determined by using one standard prurus mite positive serum and one standard negative serum and adopting a chessboard titration method. The sample concentration at which the ratio of P/N (ODpositive/ODnegative) is the largest was selected as the optimum working condition.

Dissolving the purified protein in coating solution (Na) according to optimal working concentration ₂ CO ₃ /NaHCO ₃ ) After medium sensitization, a 96-well ELISA plate (100. mu.L/well) was added thereto, and PBS-T (137mM NaCl,2.7mM KCl,10mM Na) was used after overnight incubation at 4 ℃ ₂ HPO ₄ ,2mM KH ₂ PO ₄ 0.1% v/vtween-20, pH 7.4) 3 times. Excess blocking buffer (5% skim milk powder) was added and the reaction was allowed to proceed for 1.5h at 37 ℃. Meanwhile, the serum of animals was diluted at 1:200, and after the ELISA plate was washed three times, 100. mu.L of the diluted serum was added to each well, reacted at 37 ℃ for 1 hour, and washed 3 times with PBS-T. HRP-labeled goat anti-rabbit IgG was diluted with PBS as a secondary antibody at a ratio of 1:3000, and 100. mu.L of an aliquot was added to each well, reacted at 37 ℃ for 1 hour, and washed 4 times with PBS-T. 100 μ L of LTMB staining solution (TIANGEN, Beijing, China) was added to each well, and incubated at 37 ℃ for 15 min. Add 100. mu.L of 2M H to each well ₂ SO ₄ The reaction was terminated. Taking OD on enzyme-linked immunosorbent assay ₄₅₀ Absorbance values the data were analyzed. Protein and serum working concentrations were judged by P/N (positive/negative) values.

The reagents involved in the indirect ELISA method according to this example constitute a kit.

2. Conditions for indirect ELISA and determination of cut-off values

The conditions of the indirect ELISA were determined by checkerboard titration: the optimal coating concentration for rPCys was 8.33. mu.g/mL, serum dilutions 1:200, and the dilution ratio of the secondary antibody is 1: 3000.

Screening 24 parts of serum from 48 parts of healthy rabbit for OD ₄₅₀ Determination of the values (Table 3), calculation of the Cut-off value the OD of 24 negative sera ₄₅₀ The average value is calculated by adding three times of standard deviation, and the cut-off value is 0.358 according to the formula.

TABLE 3 determination of cut-off values for rPCys indirect ELISA method

Note: if the OD450 is more than or equal to the cut-off value, the value is judged to be positive; and if the OD450 is less than the Cut-off value, judging the test result to be negative.

3. Determination of specificity and sensitivity

Tests were performed according to the optimized test conditions in 2, using 48 s of itch mite-positive serum collected and stored in the laboratory to test sensitivity, and 48 s of negative serum to determine specificity. Positive (OD450 value is more than or equal to cut-off value)/48 x 100%; specificity ═ negative (OD450 value < cut-off)/48 × 100%. Cross-reactivity assays were performed on 20 rabbit positive sera infected with sarcoptidosis and 20 rabbit positive sera infected with coccidiosis.

The sensitivity of rPCys indirect ELISA method determined according to the obtained cut-off value is 93.75% (45/48), the specificity is 93.75% (45/48), and the OD is ₄₅₀ The values are shown in the graph (Table 4-5, FIG. 4) and the cross-reactive serum profile is shown in the graph (Table 6, FIG. 4), wherein the serum-positive detection rate of the rabbit infected with Eimeria schneideri is 5% (1/20) and the serum-positive detection rate of the rabbit infected with sarcoptidosis is 85% (17/20).

TABLE 4 sensitivity of rPCys Indirect ELISA method

TABLE 5 specificity of rPCys Indirect ELISA method

TABLE 6 rPCys Indirect ELISA for detection of Ods in other parasitosis-positive rabbit serum samples ₄₅₀ Value of

The presence of cross-reactivity between the serum of itch mite and itch mite has been reported in several documents (ref: 1, Gu XB, Gu J, Ren YJ, et al. evaluation of an induced ELISA using recombinant aromatic kinase for serous insect of Psoroptesovies var. cuniculus infection in rabbites [ J ]. Front vector Sci,2019,6: 411; 2, Matthes HF, Harrison GBL, Shaw RJ, HeathaCG, Pfeffera, high T H. Crossected antibodies from Sarcoptes Susis, Chorioptes bovius and Notoeded i and anti-P. serum IgE in a Front induced physiological strain of P.12. J.22. J.: X.23. sub.12. J.23. X.12. sub.12. sub.7. J.23. sub.7. X.12. sub.7. J.23. X.23. sub.7. the cross-reactivity of itch mite and itch mite, X. sub.26. sub.12. sub.7. the same No. 3. sub.7. the same was used in FIGS Casais R, Mill a n J, Rosell JM, Dalton KP, Prieto MJ. evaluation of an ELISA using the recombinant Ss20B3 antigen for the clinical diagnosis of Sarcoptes scabies in the field of disease and animal rubbers [ j ] v.vet Parasitok, 2015,214: 315. sup. 321), but the presently disclosed diagnostic antigens do not show a high detection rate of positive sera in both itch mite and itch mite, whereas the rPCys-iELISA of the present invention shows a detection rate of rabbit positive sera in itch mite (17/20) and 93.75% in rabbit positive sera in itch mite (45/48) and a high detection rate of both parasites, which indicates that it can be used in future tests for animal parasites with high efficiency. Therefore, the PCys protein provided by the invention can also be used as a diagnostic antigen for detecting the sarcoptic mite and/or can be applied to the preparation of the diagnostic antigen for detecting the sarcoptic mite and a kit for detecting the sarcoptic mite;

in addition, both acariasis may also be treated with the same chemical miticidal agent and have a highly effective insecticidal effect (reference 1: Panigrahi PN, GuptaA. therapeutic management of curative and systemic bacteriosis in vivo. IntasPolive (2013)14(II): 319-21.26; reference 2: Elshahawy I, Goniemy AE, ErraA. epidemic biological summary on management of rabbitis in the southern region of Egypt. Sain Malayana (2016),45(5): 745-51.). In addition, 1 part of the 20 parts of coccidiosis positive serum has cross reaction with rPCys, but the OD value of the serum is extremely lower than that of the pruritic mite positive serum (P < 0.001). In view of the high sensitivity and specificity of rPCys-iELISA, the rPCys-iELISA can be used for serodiagnosis of rabbit psoriasis and/or sarcoptidosis, and provides technical support for diagnosis and control of rabbit psoriasis and sarcoptidosis in future.

4. Repeatability of the detection assay

(1) And (3) testing repeatability in batches: and (3) taking 5 parts of serum from the same batch of coated ELISA plates, detecting under the same condition, making 3 repeated holes in each serum sample, calculating the intra-batch variation coefficient according to the reading result, and evaluating the intra-batch repeatability of ELISA.

Under the same condition, 3 repeated hole detections are carried out on each serum sample by using an enzyme label plate coated in the same batch, and the result shows that the variation coefficient is 4.97% (3.12% -6.71%) < 5%, which indicates that the established indirect ELISA method has the variation within the error range and good batch repeatability.

(2) Batch-to-batch repeatability test: and (3) taking the ELISA plates coated in different batches, detecting 5 parts of serum under the same condition, making 3 repeated holes on each serum sample, calculating the batch variation coefficient according to the reading result, and evaluating the repeatability among the ELISA batches.

Under the same condition, 3 different batches of coated enzyme label plates are used for carrying out 3 repeated hole detections on each serum sample, and the result shows that the variation coefficient is 4.46% (1.97-7.78%) < 10%, which indicates that the established indirect ELISA method has good batch repeatability within the error range of batch variation.

TABLE 7 rPCys Indirect ELISA method for in-and inter-batch reproducibility and stability testing

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Sichuan university of agriculture

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130

Claims

1. Use of a cysteine protease inhibitor of psoroptes ovis for the preparation of a diagnostic antigen for psoroptes and/or sarcoptidosis.

2. Use of a cysteine protease inhibitor of psoroptes ovis as an antigen for the manufacture of a kit for the diagnosis of psoroptes and/or sarcoptidosis.

3. The use according to claim 2, wherein the kit is an ELISA kit.

4. The use according to claim 3, wherein the ELISA kit is a kit based on an ELISA indirect method.

5. An ELISA kit for diagnosing psoriatic and/or psoriatic acariasis, comprising a solid support coated with a cysteine protease inhibitor of psoriatic sheep mite.

6. The ELISA kit of claim 5, further comprising one or more of an enzyme-labeled secondary antibody, a washing solution, a developing solution, a blocking solution, a diluting solution and a stop solution.

7. The ELISA kit of claim 6, wherein the enzyme-labeled secondary antibody is goat anti-rabbit IgG labeled with HRP.