CN111965353A

CN111965353A - Application of scrapie ovis cathepsin L and ELISA kit

Info

Publication number: CN111965353A
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: 2020-11-20
Anticipated expiration: 2040-08-18
Also published as: CN111965353B

Abstract

The invention relates to the technical field of biology, and discloses a series of related applications of sheep psoroptes cathepsin L as a psoroptes and/or sarcoptidosis diagnostic antigen, and related experimental results show that the sheep psoroptes cathepsin L can be identified by positive rabbit serum of psoroptes and sarcoptidosis, which indicates that the sheep psoroptes cathepsin L has reactogenicity; meanwhile, the recombinant cathepsin L shows higher sensitivity and specificity in an indirect ELISA method (rPOCL-iELISA) established by the recombinant cathepsin L, and various results prove that the scrapie cathepsin L can be used as a diagnostic antigen of psoriasis and/or sarcoptidosis and applied to a related detection kit.

Description

Application of scrapie ovis cathepsin L and ELISA kit

Technical Field

The invention relates to the technical field of biology, in particular to application of scrapie cathepsin L 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 psoriatic mite can be timely and accurately diagnosed to prevent the spreading of the psoriatic mite in rabbit groups, and the economic loss and the animal welfare of the rabbit raising industry can be improved.

Early scrapie infection in rabbits had no obvious clinical symptoms, and this subclinical case, lasting several weeks, became the source of infection for the population. 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 timely and accurate diagnosis of the animals infected with the psoroptes ovis is beneficial to earlier and more effective development of treatment and 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 has developed rapidly in the diagnosis of parasites today due to its advantages of high accuracy and low workload. Therefore, the ELISA method can make up the defects of the traditional etiology diagnosis method and becomes a diagnosis method for the sub-clinical cases of the psoriatic acariasis. However, no diagnostic kit for rabbit itch mite disease 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 the use of the cathepsin L (pocl) of psoroptes ovis as a diagnostic antigen of psoroptes and/or sarcoptidosis and for preparing the diagnostic antigen of psoroptes and/or sarcoptidosis, such that the cathepsin L of psoroptes ovis has high specificity and sensitivity;

the invention also aims to provide the application of the scrapie cathepsin L in the preparation of a kit for detecting the psoriasis and/or the sarcoptidosis, so that an ELISA method established by the scrapie cathepsin L shows higher 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;

cathepsin L is a lysosome of cysteine proteases that hydrolyses histones, whose enzyme precursors dissociate from the receptor complex in acidic environments and promote enzyme activation to form mature cathepsin L. The structural form of the enzyme is similar to that of the papain superfamily: there are two domains that interact to form a pocket-type active site. Cathepsin L degrades collagen, elastin, laminin and extracellular matrix. Currently, much research on cathepsin L as a candidate diagnostic antigen is being carried out in the field of parasitic diseases, such as: fasciola hepatica, toxoplasma, cysticercus, etc. However, no study on cathepsin L in psoriatic or sarcoptidosis has been found.

Herein, the scrapie ovis cathepsin l (pocl) may be non-natural, e.g. synthetic or expressed from an artificial vector (often referred to in the art as recombinant protein rPOCL). 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 scrapie ovis cathepsin L gene (POCL), and carries out molecular identification on the gene through bioinformatics analysis. The immunogenicity of the recombinant protein cathepsin L (rPOCL) is studied by immunoblotting, and an indirect ELISA method is established to evaluate the potential of the recombinant protein in rabbit itch mite disease serological diagnosis. The result shows that the POCL gene ORF frame has the total length of 396bp, the recombinant fusion protein cathepsin L which is obtained by prokaryotic expression is about 33.3kDa, and the recombinant fusion protein cathepsin L can be identified by the positive rabbit serum of the psoriatic mite, which shows that the POCL gene has reactogenicity; the optimal coating concentration of the indirect ELISA method (rPOCL-iELISA) established with recombinant cathepsin L is 8.33. mu.g/mL, the serum dilution is 1:200, the secondary antibody dilution is 1:3000, the cut-off value is 0.358, and both sensitivity and specificity are 93.75% (45/48).

Based on the test result, the invention provides the application of the scrapie cathepsin L as a diagnostic antigen of the psoriatic mite and in the preparation of the diagnostic antigen of the psoriatic mite; and provides the application of the psoroptes ovis cathepsin L in the preparation of the kit for detecting the 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 psoroptes, which comprises a solid phase carrier coated with the psoroptes ovis cathepsin L. 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 scrapie ovis cathepsin L is 8.33 mug/mL, and the carrier can be coated by a coating solution which is 0.39g of Na₂CO₃，35mM NaHCO₃And adjusting the pH value to 9.6 to obtain the product, wherein the concentration of the NaCl is 0.2M.

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 solution, developing solution, confining solution, diluent and stop solution.

The enzyme-labeled secondary antibody is preferably goat anti-rabbit IgG labeled with HRP, 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 inIn a specific embodiment of the invention, the PBS-T washing solution comprises: 137mM NaCl,2.7mM KCl,10mM Na₂HPO₄,2mM KH₂PO₄0.1% 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 Na₂HPO₄，0.27gKH₂PO₄Dissolving 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 scrapie cathepsin L as the diagnostic antigen of the psoriatic mite, and the relative experiment results show that the scrapie cathepsin L can be identified by the positive rabbit serum of the psoriatic mite, which indicates that the scrapie cathepsin L has reactogenicity; meanwhile, the recombinant cathepsin L shows higher sensitivity and specificity in an indirect ELISA method (rPOCL-iELISA) established by the recombinant cathepsin L, and various results prove that the scrapie cathepsin L can be used as a diagnostic antigen of the psoroptes ovis, particularly the rabbit psoroptes ovis and applied to a related detection kit.

Drawings

FIG. 1 shows PCR amplification of POCL; lane M represents the relative molecular mass standard of DNA, lane 1 represents POCL amplification product, and 2 represents 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-POCL double digestion product; b: lane M represents the relative molecular mass standard of DNA, and

lanes

1 and 2 represent the plasmid pET32a-POCL double cleavage product;

FIG. 3 shows an immunoblot analysis of rPOCL; 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 (+) -POCL bacterial liquid induced by IPTG and the ultrasonic disruption, the precipitate is taken and dissolved in the 2M urea to obtain the staining product of Coomassie brilliant blue, lane 3 represents the purified rPOCL protein which is subjected to SDS-PAGE electrophoresis and the staining product of Coomassie brilliant blue, and lane 4 represents the incubation of the purified rPOCL with rabbit positive serum infected with psoroptes and the staining product of DAB;

FIG. 4 shows sensitivity, specificity and cross-reactivity of the indirect ELISA for rPOCL; ordinate represents OD of serum of sample₄₅₀The values, the abscissa represents the different kinds of serum samples, the thin horizontal line represents the positive detection cut-off value (cut-off value), and the short horizontal line represents the OD of the serum sample₄₅₀Mean, ns means no significant difference, and x means very significant difference.

Detailed Description

The invention discloses application of scrapie ovis cathepsin L and an ELISA kit, and a person skilled in the art can refer to the content and appropriately improve process parameters for realization. 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 invention amplifies the scrapie ovis cathepsin L coded sequence 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 an enzyme digestion connection mode, and prokaryotic expression is carried out by using escherichia coli to obtain the recombinant rPOCL.

In experiments of a specific embodiment, all experimental animals were treated strictly according to the "animal protection law of the people's republic of china" (draft published on 9/18 th in 2009). All procedures were carried out strictly in accordance with the "guidelines for animal Care by the animal ethics Committee of the university of Sichuan agriculture" (China, Yaan; approval No.: 2013-. 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 mites in sheep (reference document: old Shing, Lianhongjun, Xuxuxueping, and the like; periodic observation of development of the itch mites in sheep and biological characteristic research [ 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 ova, any clinical diseases and the epidemic history of psoriatic acariasis; other parasitic disease positive sera: the scabies mite rabbit positive serum and the rabbit coccidian positive serum are both provided by the parasite disease research center of Sichuan university of agriculture (New Zealand rabbits with single parasite pathogen infected by both rabbit sera).

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 scrapie ovis cathepsin L and an ELISA kit provided by the present invention 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)^TMRT reagent kit with DNA Eraser (Perfect Real Time)). Using touchdown PCR amplification method (table 1), the complete ORF frame sequence of POCL 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, ddH₂O 3.5μL，2×PCR Mixture5μL。

TABLE 1 PCR amplification conditions for POCL genes

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 a 200 bp-500 bp interval, the size is in line with 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; protein structure was predicted using SWISS-MODEL (http:// swissmodel. expasy. org /).

The POCL 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 total molecular mass of POCL is about 15.3kDa, the theoretical isoelectric point is 5.35, there is no transmembrane helix region, there is no signal peptide, 56.49% α -helix, 12.98% β -sheet, 5.34% β -turn and 25.19% random coil, where the amino acid sequence from position 70 to 120 is a specific cathepsin L tag. POCL has typical cathepsin L conserved functional sites, similar to those of house dust mite, european mite and mange mite.

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: rPOCL proteins were separated using 12% SDS-PAGE and transferred to nitrocellulose filter (NC membrane); sealing with skimmed milk powder for 2 hr; adding primary antibody (positive serum of itch mite disease rabbit) diluted by PBS according to the ratio of 1:100 in advance, and incubating overnight at 4 ℃; washing with TBST for 5min 3 times; adding a secondary antibody (HRP-labeled goat anti-rabbit IgG) which is diluted by PBS according to 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 bands appeared.

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

The plasmid of pET32a-POCL 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 POCL 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 rPOCL 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 concentration₂CO₃/NaHCO₃) 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 ℃₂HPO₄,2mM KH₂PO₄0.1% 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. mu.L of TMB developing solution (TIANGEN, Beijing, China) was added to each well, and incubated at 37 ℃ for 15 min. Add 100. mu.L of 2M H per well₂SO₄The reaction was terminated. Taking OD on enzyme-linked immunosorbent assay₄₅₀The absorbance values were analyzed for data. Protein and serum working concentrations were judged by P/N (positive/negative) values.

The reagents involved in the indirect ELISA method according to this embodiment 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 of rPOCL was 8.33 μ g/mL, serum dilution 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 OD₄₅₀Determination of the values (Table 3), calculation of the Cut-off value the OD of 24 negative sera₄₅₀The 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 value by rPOCL indirect ELISA method

Note: if the OD450 is more than or equal to the cut-off value, the result 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 the rPOCL indirect ELISA method was determined to be 93.75% (45/48), the specificity 93.75% (45/48), OD was determined from the cut-off value obtained₄₅₀The 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 rabbit infected with Eimeria sieboldii is 5% (1/20) and the serum-positive detection rate of rabbit infected with sarcoptidosis is 85% (17/20).

TABLE 4 sensitivity of the rPOCL indirect ELISA method

TABLE 5 specificity of the rPOCL indirect ELISA method

TABLE 6 rPOCL Indirect ELISA for detection of Od in other parasitosis-positive rabbit serum samples₄₅₀Value 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 expression enzymes for serodiagnosis of Psoroptesovies var. cuniculus infection in rabbites [ J ]. Front vector Sci,2019,6: 411; 2, Matthes HF, Harrison GBL, Shaw RJ, Heath ACG, Pfeffer A, high T H. Crossness antibodies to proteins Sactios Suis, 201oriopsis bovius and endogenous expression i and antisense P. IgE. is serum from short induced tissue culture and antigen J.358. respond to protein J.23. X.12. J. (see J.23. X.23. X.12. J.: Woob. X.23. J.23. X.23. sub.7. J.23. X.7. sub.7. the cross-reaction between itch mite and itch mite J.26. the serum of psorales. the same No. 7. the same No. 5. 2. the same as the serum of the same The detection rate of the rPOCL-iELISA for rabbit positive serum of itch mite and scab mite is 85% (17/20) and 93.75% (45/48) and has higher detection efficiency for both parasitic diseases, which indicates that the rPOCL-iELISA can be efficiently used in animal parasitic disease detection in future work. Therefore, the POCL 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 diseases can also be treated with the same chemical miticidal agent and have a high insecticidal effect (reference 1: Panigahi PN, Gupta A. therapeutic management of current and systemic availability in nanoparticles 14(II): 319-21.26; reference 2: Elshahawy I, Goniemy AE, Erraa A. environmental survival on limit of nanoparticles in the southern areas of Egypt. Sains Malaysian (2016),45(5): 745-51). In addition, 1 part of the 20 parts of coccidiosis positive serum has cross reaction with rPOCL, but the OD value is extremely lower than that of the psoriatic mite positive serum (P < 0.001). In view of the high sensitivity and specificity of the rPOCL-iELISA, the invention considers that the rPOCL-iELISA can be used for serological diagnosis of rabbit psoriasis and/or sarcoptidosis, and provides technical support for diagnosis and control of rabbit psoriasis and sarcoptidosis in the future.

4. Repeatability of the detection assay

(1) And (3) carrying out batch repeatability inspection: and (3) taking 5 parts of serum from the coated ELISA plate of the same batch, 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) And (3) 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 rPOCL Indirect ELISA method for in-batch and inter-batch repeatability 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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Claims

1. The sheep psoroptes cathepsin L is used as a diagnostic antigen of psoroptes and/or sarcoptidosis or used for preparing the diagnostic antigen of the psoroptes and/or sarcoptidosis.

2. Application of the scrapie ovis cathepsin L in preparation of a kit for diagnosing the psoriasis and/or the 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 psoriasis and/or sarcoptidosis, which is characterized by comprising a solid phase carrier coated with psoriasis ovis cathepsin L.

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.