Specific diagnostic antigen for canine clonorchiasis and its ELISA diagnostic kit

Abstract

The present invention relates to a specific diagnostic antigen for canine clonorchiasis and an ELISA diagnostic kit thereof. The antigen comprises two proteins, namely HP80 with an amino acid sequence as shown in SEQ ID No:1 and HP318 with an amino acid sequence as shown in SEQ ID No:2, both of which are excretory and secretory antigens of canine clonorchiasis. HP80 or HP318 is used to establish an indirect ELISA method for detecting canine clonorchiasis. The detection kit has strong specificity, high sensitivity, good repeatability, is simple and fast, and is suitable for application in aspects such as large-scale epidemiological surveys. In addition, the present invention is non-toxic to operators and non-polluting to the environment, and has broad market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biological immune detection, and in particular to a canine clonorchiasis-specific diagnostic antigen and an ELISA diagnostic kit thereof. Background Art

[0002] Clonorchis sinensis belongs to the genus Clonorchis of the family Opisthorchidae. It parasitizes in the hepatobiliary and gallbladder of many mammals, including dogs, cats and humans. It is an important foodborne zoonosis parasite. Dogs become sick from eating raw or half-cooked fish and shrimp containing infectious cysts of Clonorchis sinensis. The cysts are excysted in the duodenum, and under the chemotaxis of bile, the larvae migrate into the hepatobiliary. The migration of the worm not only causes damage to the bile duct, but also releases excretory and secretory antigens that can stimulate the hepatobiliary and induce inflammation. Sick dogs show malnutrition, diarrhea, jaundice, and in severe cases, death. Dogs are the natural hosts of the disease. Adult worms can survive in the hepatobiliary of dogs for several years. Sick dogs are an important source of infection.

[0003] Clonorchis sinensis is hermaphroditic. Dogs can mature and ovulate in large numbers 21 days after infection, and sick dogs themselves become sources of infection. Currently, there is no effective commercial vaccine on the market to prevent canine clonorchiasis. Therefore, accurate and rapid diagnosis and treatment of sick dogs to prevent further spread of the pathogen is a prerequisite for efficient prevention and control of clonorchiasis sinensis.

[0004] The internationally recognized diagnostic method is to detect eggs in feces, but this method has disadvantages: (1) it is easy to miss when the infection is mild; (2) it requires professional personnel to operate, which is time-consuming; (3) it is not suitable for large-scale testing, etc. Clinically, there is an urgent need for antigens with strong specificity and high sensitivity for immediate detection of the disease, so as to achieve the goal of early detection and early treatment, and effectively prevent and control clonorchiasis. Summary of the invention

[0005] The purpose of the present invention is to disclose a canine clonorchiasis-specific diagnostic antigen and an ELISA diagnostic kit thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0006] The first aspect of the present invention is to provide a canine Clonorchiasis-specific antigen.

[0007] The second aspect of the present invention is to provide two gene fragments encoding canine clonorchiasis-specific antigens.

[0008] The third aspect of the present invention is to provide a recombinant expression vector containing the gene fragment described in the second aspect of the present invention.

[0009] The fourth aspect of the present invention is to provide a host bacteria containing the recombinant expression vector described in the third aspect of the present invention.

[0010] The fifth aspect of the present invention is to provide a method for preparing the canine clonorchiasis-specific antigen described in the first aspect of the present invention.

[0011] The sixth aspect of the present invention is to provide a kit containing the canine clonorchiasis-specific antigen described in the first aspect of the present invention.

[0012] The seventh aspect of the present invention is to provide the use of the canine clonorchiasis-specific antigen described in the first aspect of the present invention in the preparation of a drug for diagnosing, treating or preventing canine clonorchiasis.

[0013] The canine clonorchiasis-specific antigen described in the first aspect of the present invention comprises any one of the following proteins (A)-(C):

[0014] (A) a protein with an amino acid sequence as shown in SEQ ID No: 1 or SEQ ID No: 2;

[0015] (B) a protein having the same function as the amino acid sequence shown in SEQ ID No: 1 or SEQ ID No: 2, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0016] (C) A protein having 99% or more, 95% or more, 90% or more, 85% or more or 80% homology with the amino acid sequence defined in any one of (A) or (B) and having the same function.

[0017] The protein with the amino acid sequence shown in SEQ ID No: 1 is named HP80, and the protein with the amino acid sequence shown in SEQ ID No: 2 is named HP318, both of which are excretory and secretory antigens of Clonorchis sinensis. Excretory and secretory antigens are metabolites released by parasites during the parasitic period in the host, and their components are complex, including proteins, lipids and other small molecules. Excretory and secretory antigens have good reactogenicity and can cause a strong immune response in the body. In addition, the antigenic substances can be released to the body in the early stage of parasitic infection, triggering an immune response and causing damage to the body.

[0018] "Homology" as used herein refers to the correlation between two amino acid sequences, which is described by the parameter "Identity". Preferably, the homology of more than 95% may be at least 96%, 97%, 98%, 99% identity. The homology of more than 90% may be at least 91%, 92%, 93%, 94% identity. The homology of more than 85% may be at least 86%, 87%, 88%, 89% identity. The homology of more than 80% may be at least 81%, 82%, 83%, 84% identity. And the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived.

[0019] The gene fragments described in the second aspect of the present invention include: a gene fragment encoding HP80 as shown in SEQ ID No:3; or a gene fragment encoding HP318 as shown in SEQ ID No:4.

[0020] The recombinant expression vector of the third aspect of the present invention contains a gene fragment encoding HP80 or HP318. The gene fragment shown in SEQ ID No: 3 is used as a template, and PCR amplification is performed by a primer pair shown in SEQ ID No: 5 and SEQ ID No: 6; or the gene fragment shown in SEQ ID No: 4 is used as a template, and PCR amplification is performed by a primer pair shown in SEQ ID No: 7 and SEQ ID No: 8. After PCR amplification, an EcoR I restriction site is added to the 5' end of the amplified product, and an Xho I restriction site is added to the 3' end. The amplified product is connected to a double-enzyme-digested vector plasmid to obtain the recombinant expression vector.

[0021] The host bacteria of the fourth aspect of the present invention contain the recombinant expression vector of the third aspect of the present invention. The recombinant expression vector is first transfected into monoclonal bacteria, the bacteria are lysed after expansion culture, the cloned recombinant expression vector is extracted, and then transferred into the expression bacteria.

[0022] The method for preparing the canine clonorchiasis-specific antigen according to the fifth aspect of the present invention comprises the following steps:

[0023] 1) constructing the host bacteria described in the fourth aspect of the present invention;

[0024] 2) expanding the host bacteria, breaking the bacteria after inducing expression, centrifuging, and collecting the supernatant;

[0025] 3) After purification, the canine clonorchiasis-specific antigen is obtained.

[0026] The kit described in the sixth aspect of the present invention contains the canine clonorchiasis-specific antigen described in the first aspect of the present invention. The canine clonorchiasis-specific antigen can be used to construct an indirect ELISA method to detect whether there is an antibody in a serum sample, thereby determining whether a dog has canine clonorchiasis.

[0027] In some application embodiments of the sixth aspect of the present invention, it also includes a 96-well ELISA reaction plate, and at least one of standard negative serum, standard positive serum, diluent, blocking solution, washing solution, enzyme-labeled secondary antibody, color developing solution or stopping solution.

[0028] Furthermore, the canine clonorchiasis-specific antigen is coated and fixed on the 96-well ELISA reaction plate. The canine clonorchiasis-specific antigen coating concentration is 0.5 μg / well, and the coating solution is a PBS solution. The PBS solution is composed of: 9g sodium chloride, 0.2g potassium chloride, 1.42g disodium hydrogen phosphate, 0.27g potassium dihydrogen phosphate, dissolved in 800mL deionized water, fixed to 1L, and adjusted to pH 7.4.

[0029] In some application embodiments of the sixth aspect of the present invention, the blocking solution is 5% skim milk; the sample diluent is phosphate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled rabbit anti-dog IgG secondary antibody; the color developing solution is a single-component TMB color developing solution; and the stop solution is a sulfuric acid solution with a concentration of 2M. The PBST washing solution is composed of: 500μLTween-20 is added to 1L PBS solution.

[0030] The beneficial effect of the present invention is that an indirect ELISA method for detecting canine clonorchiasis is established by using HP80 or HP318. The detection kit has strong specificity, high sensitivity, good repeatability, is simple and fast, and is suitable for large-scale epidemiological surveys and other applications. In addition, the present invention is non-toxic to operators and does not pollute the environment, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a photo of Clonorchis sinensis obtained in Example 1;

[0032] Figure 2 is the electrophoresis diagram of HP80 in Example 2;

[0033] Figure 3 is the electrophoresis image of HP318 in Example 3;

[0034] Figure 4 This is a graph showing the serum reactivity of HP80 to Clonorchis sinensis at different stages in Example 4;

[0035] Figure 5 This is a graph showing the serum reactivity of HP318 to Clonorchis sinensis at different stages in Example 4;

[0036] Figure 6 It is the HP80 kit specific analysis in Example 5;

[0037] Figure 7 It is the HP318 kit specific analysis in Example 5;

[0038] Figure 8 It is the sensitivity test of HP80 test kit in Example 5;

[0039] Fig. 9 This is the sensitivity test of the HP318 test kit in Example 5. DETAILED DESCRIPTION

[0040] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0041] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0042] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the kits and product instructions; the biological materials of the kits, unless otherwise specified, can be obtained from commercial channels.

[0043] Example 1: Establishment of Clonorchis sinensis infection model in dogs

[0044] Naturally infected mullets were collected from the endemic area of ​​Clonorchis sinensis in Changchun, Jilin Province [ Figure 1 [As shown in (A)] The head, tail, fins and internal organs of the wheat ear fish were removed, the muscle tissue was cut into pieces, and the muscle tissue was digested with artificial digestive fluid (8g pepsin, 10mL hydrochloric acid, 9g sodium chloride, fixed to 1L) at 37°C for 5 hours. Undigested tissue was removed by filtration, and the mixture was allowed to stand for 20 minutes. The supernatant was discarded, and physiological saline was added to the precipitate, and the mixture was precipitated again for 20 minutes. This was repeated four times. After the last digestion, the precipitate was collected and the cysts of Clonorchis sinensis were picked out under a microscope [as shown in Figure 1 ] 6-8 week old beagle dogs were orally infected with 500 viable metacercariae. Serum was collected on days 0, 7, 14, 21, 28, and 35 of infection, separated by centrifugation (3500 rpm for 10 min), and stored at -80°C for further use. 77 days after infection, the dogs were euthanized, and the livers were removed to collect adult worms of C. sinensis [as shown in Figure 2A]. Figure 1 (as shown in (C)].

[0045] Example 2: Preparation of canine clonorchiasis-specific diagnostic antigen HP80

[0046] 1. According to the gene sequence of HP80 (SEQ ID No: 3), design amplification primers:

[0047] HP80F: 5'- GAATTC TCAAAGCAGAGGAAGC-3' (SEQ ID No: 5), the underline is the EcoR I restriction site;

[0048] HP80R: 5'- CTCGAG ATATCTATATTGAGCTGT-3' (SEQ ID No: 6), the underline indicates the Xho I restriction site.

[0049] 2. Amplify the HP80 gene fragment with restriction endonuclease sites by PCR.

[0050] PCR reaction system: 12.5 μL of 2×PCR Mix Taq enzyme, 2 μL of HP80 cDNA template, 1 μL each of upstream primer HP80F and downstream primer HP80R, add water to 25 μL.

[0051] PCR amplification program: 94℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 52℃ annealing for 30 seconds, 72℃ extension for 1 minute, a total of 32 cycles; final extension at 72℃ for 8 minutes. PCR products were identified by 2% agarose gel electrophoresis. The results are as follows Figure 2 As shown in (A).

[0052] 3. Construct the recombinant expression vector HP80-pET-32.

[0053] According to the instructions of the DNA agarose gel recovery kit of Tiangen Biotechnology Co., Ltd., the PCR product was recovered, and the PCR product and the Pet-32a vector plasmid were double-digested with restriction endonucleases BamH I and Xho I. The digestion system is: 1 μL of BamH I and Xho I, 2 μL of 10× buffer, and the plasmid is added to make up to 20 μL. After mixing, it was placed in a 37°C water bath for constant temperature digestion for 2 hours. The digestion product was subjected to 2% agarose gel electrophoresis, and the digested vector and target gene were recovered again.

[0054] The PCR product recovered by enzyme digestion was connected to the pET-32a expression vector. The connection system was as follows: 5 μL of PCR recovery product, 3 μL of pET-32a vector, 1 μL of 10×Ligation buffer, 1 μL of T4 DNA ligase. After mixing, the mixture was connected at 16°C for 12 hours.

[0055] The ligation product was mixed with DH5α competent cells at a ratio of 1:10, placed in an ice-water mixture for 30 minutes, then placed in a 42°C water bath for 90 seconds, and then continued to ice bath for 2 minutes. 500 μL of non-resistant LB culture medium was added to the mixture, and it was shaken in a 37°C shaker for 45 minutes. 100 μL of bacterial culture was evenly spread on the LB solid medium containing ampicillin resistance, and the medium was inverted in a 37°C incubator for 18 hours. A round, single white colony was picked and placed in 500 μL of ampicillin-resistant LB medium for 6 hours, and sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing.

[0056] The correctly sequenced bacterial solution was inoculated with bacteria at a ratio of 1:1000 between bacterial solution and culture medium, cultured in a 37°C incubator for 8 hours, and the plasmid was extracted according to the instructions of the Tiangen Bioplasmid Mini-Extraction Kit.

[0057] The extracted plasmid was double digested with restriction endonucleases BamHI and XhoI for identification. The digestion system was: 1μL each of BamHI and XhoI, 2μL of 10× buffer, and the plasmid was added to make up to 20μL. After mixing, it was placed in a 37℃ water bath for constant temperature digestion for 2 hours. The digestion products were analyzed by 2% agarose gel electrophoresis. The results are as follows Figure 2 As shown in (B).

[0058] 4. Identification of recombinant protein HP80.

[0059] The plasmid of the correct bacterial solution identified by sequencing and double enzyme digestion was transferred into BL21 (DE3) competent cells, spread on LB solid medium containing ampicillin resistance selection marker, and cultured in a 37°C incubator for 16 hours. Pick a round, single white colony and culture it in 500 μL ampicillin resistance LB medium for 6 hours, and send it to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing.

[0060] The correctly sequenced bacterial solution was inoculated with ampicillin-resistant culture medium at a ratio of 1:1000 and cultured in a 37°C incubator for 6 hours to reach the logarithmic growth phase. 600 When the value reached 0.6, IPTG was added to a final concentration of 1 mmol / L for induction, and the cells were cultured at 37°C for 8 hours by shaking. The cells were collected by centrifugation at 12000g for 1 minute. At the same time, the pET-32a empty vector was treated in the same way as a blank control.

[0061] Take a portion of the bacterial pellet, add an appropriate amount of RIPA lysis buffer, lyse on ice for 30 minutes, centrifuge at 12000g for 5 minutes, collect the supernatant and pellet, and add an appropriate amount of PBS to resuspend the pellet.

[0062] 5. Purification of recombinant protein.

[0063] Take 200 μL HP80-pET-32 bacterial solution and add it to 200 mL ampicillin-resistant culture medium. Incubate in a 37°C incubator for 6 hours to reach the logarithmic growth phase. OD 600When the value reaches 0.6, IPTG is added to a final concentration of 1 mmol / L for induction. After continued shaking culture at 37°C for 8 hours, centrifuge at 12000g for 5 minutes to collect the bacteria. Add PBS at 5mL / g to resuspend the bacteria, use an ultrasonic disruptor with a power of 150W to ultrasonically disrupt the bacteria, ultrasonically work for 5 seconds, with an interval of 5 seconds, for a total of 20 minutes. After completion, centrifuge at 12000g for 15 minutes and collect the supernatant. Purify the protein according to the instructions of the His tag protein purification kit, and store the collected recombinant protein at -20°C. The protein concentration after purification is 4μg / μL.

[0064] The whole bacteria, supernatant, precipitate of the induced pET-32 vector and the whole bacteria, supernatant, precipitate and purified target protein of the induced HP80-pET-32 recombinant expression vector were respectively subjected to SDS-PAGE electrophoresis. Apply a voltage of 80V for 30 minutes, then apply 110V for 1 hour, except for the bottom bromophenol blue dye part in the upper concentrated gel and the separation gel, soak in Coomassie brilliant blue for 15 minutes, and decolorize with a decolorizing solution until the background is clear. The results are as follows Figure 2 As shown in (C), lane M is a protein marker; lane 1 is the induced empty supernatant; lane 2 is the induced empty precipitate; lane 3 is the induced HP80 supernatant; lane 4 is the induced HP80 precipitate; and lane 5 is the purified HP80 protein.

[0065] Example 3: Preparation of canine Clonorchiasis-specific diagnostic antigen HP318

[0066] 1. According to the gene sequence of HP318 (SEQ ID No: 4), design amplification primers:

[0067] HP318F: 5'-GAATTCGGTCAGGGCTATGGTAAACT-3' (SEQ ID No: 7), the underline indicates the EcoR I restriction site;

[0068] HP318R: 5'-CTCGAGAGCCACCGAGGCCTGCGG-3' (SEQ ID No: 8), the underline indicates the Xho I restriction site.

[0069] 2. Amplify the HP318 gene fragment with restriction endonuclease sites by PCR.

[0070] PCR reaction system: 2×PCR Mix Taq enzyme 12.5 μL, HP318 cDNA template 2 μL, upstream primer HP318F and downstream primer HP318R 1 μL each, add water to 25 μL.

[0071] PCR amplification program: 94℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 52℃ annealing for 30 seconds, 72℃ extension for 1 minute, a total of 32 cycles; final extension at 72℃ for 8 minutes. PCR products were identified by 2% agarose gel electrophoresis. The results are as follows Figure 3 As shown in (A).

[0072] 3. Construct the recombinant expression vector HP318-pET-32.

[0073] According to the instructions of the DNA agarose gel recovery kit of Tiangen Biotechnology Co., Ltd., the PCR product was recovered, and the PCR product and the Pet-32a vector plasmid were double-digested with restriction endonucleases BamH I and Xho I. The digestion system is: 1 μL of BamH I and Xho I, 2 μL of 10× buffer, and the plasmid is added to make up to 20 μL. After mixing, it was placed in a 37°C water bath for constant temperature digestion for 2 hours. The digestion product was subjected to 2% agarose gel electrophoresis, and the digested vector and target gene were recovered again.

[0074] The PCR product recovered by enzyme digestion was connected to the pET-32a expression vector. The connection system was as follows: 5 μL of PCR recovery product, 3 μL of pET-32a vector, 1 μL of 10×Ligation buffer, 1 μL of T4 DNA ligase. After mixing, the mixture was connected at 16°C for 12 hours.

[0075] The ligation product was mixed with DH5α competent cells at a ratio of 1:10, placed in an ice-water mixture for 30 minutes, then placed in a 42°C water bath for 90 seconds, and then continued to ice bath for 2 minutes. 500 μL of non-resistant LB culture medium was added to the mixture, and it was shaken in a 37°C shaker for 45 minutes. 100 μL of bacterial culture was evenly spread on the LB solid medium containing ampicillin resistance, and the medium was inverted in a 37°C incubator for 18 hours. A round, single white colony was picked and placed in 500 μL of ampicillin-resistant LB medium for 6 hours, and sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing.

[0076] The correctly sequenced bacterial solution was inoculated with bacteria at a ratio of 1:1000 between bacterial solution and culture medium, cultured in a 37°C incubator for 8 hours, and the plasmid was extracted according to the instructions of the Tiangen Bioplasmid Mini-Extraction Kit.

[0077] The extracted plasmid was double digested with restriction endonucleases BamHI and XhoI for identification. The digestion system was: 1μL each of BamHI and XhoI, 2μL of 10× buffer, and the plasmid was added to make up to 20μL. After mixing, it was placed in a 37℃ water bath for constant temperature digestion for 2 hours. The digestion products were analyzed by 2% agarose gel electrophoresis. The results are as follows Figure 3 As shown in (B).

[0078] 4. Identification of recombinant protein HP318.

[0079] The plasmid of the correct bacterial solution identified by sequencing and double enzyme digestion was transferred into BL21 (DE3) competent cells, spread on LB solid medium containing ampicillin resistance selection marker, and cultured in a 37°C incubator for 16 hours. Pick a round, single white colony and culture it in 500 μL ampicillin resistance LB medium for 6 hours, and send it to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing.

[0080] The correctly sequenced bacterial solution was inoculated with ampicillin-resistant culture medium at a ratio of 1:1000 and cultured in a 37°C incubator for 6 hours to reach the logarithmic growth phase. 600 When the value reached 0.6, IPTG was added to a final concentration of 1 mmol / L for induction, and the cells were cultured at 37°C for 8 hours by shaking. The cells were collected by centrifugation at 12000g for 1 minute. At the same time, the pET-32a empty vector was treated in the same way as a blank control.

[0081] Take a portion of the bacterial pellet, add an appropriate amount of RIPA lysis buffer, lyse on ice for 30 minutes, centrifuge at 12000g for 5 minutes, collect the supernatant and pellet, and add an appropriate amount of PBS to resuspend the pellet.

[0082] 5. Purification of recombinant protein.

[0083] Take 200 μL HP318-pET-32 bacterial solution and add it to 200 mL ampicillin-resistant culture medium. Incubate in a 37°C incubator for 6 hours to reach the logarithmic growth phase. OD 600 When the value reaches 0.6, IPTG is added to a final concentration of 1 mmol / L for induction. After continued shaking culture at 37°C for 8 hours, centrifuge at 12000g for 5 minutes to collect the bacteria. Add PBS at 5mL / g to resuspend the bacteria, use an ultrasonic disruptor with a power of 150W to ultrasonically disrupt the bacteria, ultrasonically work for 5 seconds, with an interval of 5 seconds, for a total of 20 minutes. After completion, centrifuge at 12000g for 15 minutes and collect the supernatant. Purify the protein according to the instructions of the His tag protein purification kit, and store the collected recombinant protein at -20°C. The protein concentration after purification is 4μg / μL.

[0084] The whole bacteria, supernatant, precipitate of the induced pET-32 vector and the whole bacteria, supernatant, precipitate and purified target protein of the induced HP318-pET-32 recombinant expression vector were respectively subjected to SDS-PAGE electrophoresis. Apply a voltage of 80V for 30 minutes, then apply 110V for 1 hour, except for the bottom bromophenol blue dye part in the upper concentrated gel and the separation gel, soak in Coomassie brilliant blue for 15 minutes, and decolorize with a decolorizing solution until the background is clear. The results are as follows Figure 3As shown in (C), lane M is a protein marker; lane 1 is the induced empty supernatant; lane 2 is the induced empty precipitate; lane 3 is the induced HP318 supernatant; lane 4 is the induced HP318 precipitate; and lane 5 is the purified HP318 protein.

[0085] Example 4: Reactogenicity analysis of recombinant proteins

[0086] The purified recombinant proteins (HP80 or HP318) were used for western blot experiments. The positive sera of different infections with Clonorchis sinensis were used as primary antibodies, and HRP-labeled rabbit anti-dog IgG was used as secondary antibodies to detect the reactivity of the two proteins to dog sera. The specific experiments are as follows:

[0087] 1) Take 10 μg of recombinant protein and run SDS-PAGE electrophoresis. First, apply a voltage of 80 V for 30 minutes, then apply a voltage of 110 V for 1 hour. Cut off the upper layer of concentrated gel and the bottom layer of bromophenol blue dye in the separation gel, soak them in water for later use.

[0088] 2) Cut filter paper and cellulose acetate membrane slightly larger than the protein gel, and soak them in the transfer solution for 1 minute.

[0089] 3) Use a semi-dry transfer instrument to transfer the membrane. Place the filter paper, membrane, protein gel, and filter paper in order to avoid bubbles. Set the parameters to 15V and 20 minutes.

[0090] 4) After the transfer, the membrane was blocked with 5% skim milk at 4°C for 12 hours, and then rinsed with PBST three times, each time for 5 minutes.

[0091] 5) Incubation with primary antibody. Serum from canine Clonorchis sinensis infection at 0, 7, 14, 21, and 28 days was diluted with PBST at 1:200, incubated at 37°C for 1 hour, and rinsed with PBST three times, 5 minutes each time.

[0092] 6) Incubate with secondary antibody: Dilute HRP-labeled rabbit anti-dog IgG (secondary antibody) at 1:10,000 in PBST, incubate at 37°C for 1 hour, and rinse three times.

[0093] 7) Color development: Prepare the working color development solution according to the DAB instructions, immerse the membrane in the color development solution, and incubate at 37°C in the dark for 10 minutes.

[0094] Results: Figure 4 As shown in the figure, HP80 protein can identify positive sera on days 21, 28, and 35 after dog infection, indicating that HP80 protein has the potential to become an antigen for diagnosing canine clonorchiasis; Figure 5 As shown, HP318 protein can identify positive sera on days 21, 28, and 35 after dog infection, indicating that HP318 protein has the potential to become an antigen for diagnosing canine Clonorchiasis sinensis.

[0095] Example 5: Establishment of ELISA

[0096] 1. Indirect ELISA method for detecting antibodies in serum

[0097] The recombinant protein (HP80 or HP318) was used to coat the ELISA plate to detect the positive and negative serum of canine clonorchiasis and to verify the detection effect of the recombinant protein. The specific steps are as follows:

[0098] 1) Coating. Use PBS as the coating solution, dilute the recombinant protein to 10 μg / mL, add 200 μL per well, and coat at 4°C for 12 hours. Shake off the coating solution in the wells, add 200 μL PBST to rinse three times, and pat dry for the last time.

[0099] 2) Blocking: PBST containing 5% skim milk was used as the blocking solution, 200 μL per well, and after blocking at 4°C for 12 hours, the coating solution in the well was shaken dry, and 200 μL PBST was added to rinse three times, and patted dry for the last time.

[0100] 3) Incubate with primary antibody. Dilute the primary antibody with PBST at 1:200, add positive and negative sera of Clonorchis sinensis respectively, and repeat three times. Incubate at 37℃ for 2 hours, shake off the coating solution in the wells, add 200μL PBST to rinse three times, and pat dry for the last time.

[0101] 4) Incubate with secondary antibody. Dilute the secondary antibody with PBST at 1:10000, incubate at 37℃ for 1 hour, spin dry the coating solution in the wells, add 200μL PBST to rinse three times, and pat dry for the last time.

[0102] 5) Color development: Add 50 μL of TMB color development solution to each well and develop the color at 37°C in the dark for 10 minutes.

[0103] 6) Stop: Add 50 μL of stop solution to each well.

[0104] 7) Read the readings. Use an OD 450 Read the value.

[0105] 2. Checkerboard method to determine antigen coating concentration and antibody dilution multiple

[0106] The antigen coating concentration and antibody dilution multiple were determined by the chessboard method, and the recombinant protein was coated at 20, 10, 5, and 2.5 μg / mL. The positive and negative serum of C. sinensis were diluted at 1:20, 1:40, 1:80, 1:100, and 1:200, respectively, and the OD 450 After the values ​​were calculated, the condition with the largest P / N value was selected to determine the dilution multiples of the antigen and antibody. The results are shown in Tables 1 and 2.

[0107] Table 1. Test results of HP80 checkerboard method

[0108]

[0109] Table 2. Test results of HP318 checkerboard method

[0110]

[0111] When the HP80 protein was coated at 10 μg / mL and the serum dilution factor was 1:40, the P / N value was the largest. When the HP318 protein was coated at 5 μg / mL and the serum dilution factor was 1:80, the P / N value was the largest.

[0112] 3. Determination of the positive and negative critical values

[0113] Using HP80 to detect 52 canine negative sera, calculate the S / P value, (S / P = (sample OD 450nm value - negative control OD 450nm mean) / (positive control OD 450nm mean - negative control OD 450nm mean)). The average value X of the serum samples was 0.02, and the standard deviation SD was 0.029. Taking X + 3SD as the critical value, if the serum sample S / P ≥ X + 3SD, it was judged as positive; if S / P < X + 3SD, it was judged as negative. That is, when the serum sample S / P ≥ 0.107, it was positive; when S / P < 0.107, it was negative.

[0114] Using HP318 to detect 52 canine negative sera, calculate the S / P value. The average value X of the serum samples was 0.035, and the standard deviation SD was 0.046. Taking X + 3SD as the critical value, if the serum sample S / P ≥ X + 3SD, it was judged as positive; if S / P < X + 3SD, it was judged as negative. That is, when the serum sample S / P ≥ 0.173, it was positive; when S / P < 0.173, it was negative.

[0115] 4. Specificity detection

[0116] According to the indirect ELISA method, two recombinant proteins (HP80 or HP318) were used to detect positive serum samples of Clonorchis sinensis, Metorchis orientalis, Toxocara canis, Dirofilaria immitis, Strongyloides stercoralis, Dipylidium caninum, Echinococcus granulosus, Babesia and Toxoplasma gondii in dogs respectively. The detection results of the HP80 protein are as Figure 6 shown, and the detection results of the HP318 protein are as Figure 7 shown. Neither of them showed cross-reactivity with positive serum of other types of canine parasites, indicating strong specificity.

[0117] 5. Sensitivity detection

[0118] To test the sensitivity of the two recombinant proteins, the standard positive serum was diluted 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, and 1:500, respectively. Figure 8 and Fig. 9 As shown, the sensitivity of HP80 protein to the Cstr1 antigen polypeptide specific to Clonorchis sinensis was 1:400, and the sensitivity of HP318 protein to the Cstr1 antigen polypeptide specific to Clonorchis sinensis was 1:350.

[0119] 6. Repeatability Experiment

[0120] The same batch of ELISA plates and different batches of coated ELISA plates were used to test 10 sera (5 positive and 5 negative). Each serum was repeated twice, and the inter-batch and intra-batch tests were performed according to the optimized ELISA method. The coefficient of variation was calculated according to the formula CV (%) = SD / X-×100%, and the inter-batch repeatability was tested. The results are shown in Tables 3 and 4.

[0121] Table 3. HP80 repeatability test results

[0122]

[0123] Table 4. HP318 repeatability test results

[0124]

[0125]

[0126] It can be seen from Tables 3 and 4 that the coefficients of variation between and within batches were less than 10%, and the experimental repeatability was good.

[0127] 7. Clinical sample testing

[0128] According to the optimized indirect ELISA method, the two recombinant proteins were used to detect 78 canine clinical serum samples of Clonorchis sinensis in the endemic area of ​​Clonorchis sinensis, and the detection was compared with the gold standard fecal microscopy. The test results are shown in Table 5.

[0129] Table 5. Clinical test results

[0130]

[0131] 33 serum samples tested by HP80 had S / P values ​​greater than the critical value (0.107), which were positive for Clonorchis sinensis, with a positive rate of 42.31%. 34 samples were positive by fecal microscopy, with a positive rate of 43.59%. Compared with the gold standard, the sensitivity of the indirect ELISA established in this study was 97.06% and the specificity was 100%. 32 serum samples tested by HP80 had S / P values ​​greater than the critical value, which were positive for Clonorchis sinensis, with a positive rate of 42.31%. 34 samples were positive by fecal microscopy, with a positive rate of 41.03%. Compared with the gold standard, the sensitivity of the indirect ELISA established in this study was 94.18% and the specificity was 100%.

[0132] The kit for canine clonorchiasis of the present invention has the advantages of strong specificity, high sensitivity, accurate detection results, etc., and the method is simple to operate, suitable for large-scale screening of canine clonorchiasis at the grassroots level, and provides technical support for the diagnosis and prevention and control of the disease.

[0133] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. Use of an antigen having an amino acid sequence as shown in SEQ ID No: 1 in the preparation of a drug for diagnosing canine clonorchiasis.

2. Use of an antigen having an amino acid sequence as shown in SEQ ID No: 2 in the preparation of a drug for diagnosing canine clonorchiasis.

3. An ELISA kit for diagnosing canine clonorchiasis, characterized in that: It includes an antigen whose amino acid sequence is shown as SEQ ID No: 1 and / or SEQ ID No: 2, and also includes a 96-well ELISA reaction plate, and at least one of a standard negative serum, a standard positive serum, a diluent, a blocking solution, a washing solution, an enzyme-labeled secondary antibody, a color developing solution or a stopping solution.

4. The kit according to claim 3, characterized in that: The 96-well ELISA reaction plate is coated with the antigen.

5. The kit according to claim 3, characterized in that: The blocking solution is 5% skim milk; the diluent is phosphate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled rabbit anti-dog IgG secondary antibody; the color developing solution is a single-component TMB color developing solution; and the stop solution is a 2 M sulfuric acid solution.

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