A spn1-1 protein fragment and application thereof in human hydatidosis

Abstract

The application discloses a SPON1-1 protein fragment and application thereof in human echinococcosis. The polypeptide comprises one or more of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The recombinant protein comprises the amino acid sequence shown in SEQ ID NO:7; the mutated amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, 99% identity with the amino acid sequence of the recombinant protein, and maintains the function of the recombinant protein. The application further discloses application of the polypeptide or the recombinant protein or a mutation thereof or the marker combination in preparation of an antibody against echinococcosis or a diagnostic agent for diseases caused by echinococcosis granularis. The new antigen SPON1-1 of echinococcosis can be used to prepare an ELISA kit and is used for clinical detection of human echinococcosis; and can be purified in a large amount, so that large-scale screening of human echinococcosis is facilitated.

Description

Technical Field

[0001] This invention relates to the field of immunomedicine, and in particular to a polypeptide, namely a fragment of the human echinococcosis neoantigen SPON1-1 protein, a recombinant protein or a mutation thereof, a nucleic acid sequence thereof, and its application in the preparation of drugs for the detection or treatment of human echinococcosis. Background Technology

[0002] Echinococcosis, also known as hydatid disease, is a zoonotic disease. Human echinococcosis is mainly divided into cystic echinococcosis and alveolar echinococcosis. Imaging is the most direct and commonly used diagnostic method for echinococcosis. However, due to the long incubation period of echinococcosis and the fact that the cysts formed by the echinococcosis can only be detected by imaging when they reach a certain size, serological testing is one of the important auxiliary means for diagnosing echinococcosis through imaging. Currently, the main serological diagnostic antigens that can be used to diagnose echinococcosis are Antigen B and Antigen 5, as well as some recombinant antigens related to these two antigens.

[0003] Extracting and identifying echinococcosis proteins from echinococcosis cysts isolated from patients after surgery is technically challenging. If the number of identified echinococcosis proteins is limited, selecting proteins that may serve as echinococcosis antigens becomes even more difficult.

[0004] Secondly, there are few single antigen types. Currently, the most widely used diagnostic antigens for echinococcosis are Antigen B and Antigen 5, and many studies have shown that these two antigens can produce a certain number of false positives or false negatives in serum testing.

[0005] Commercially available echinococcosis antigen is a naturally purified antigen, isolated and purified from soluble antigen fragments after the fragmentation of Echinococcus granulosus larvae. It is a mixture containing multiple echinococcosis proteins. The more proteins mixed in, the more likely it is to produce false negative or false positive results in serum testing. Summary of the Invention

[0006] To address the technical shortcomings of existing technologies, such as difficulties in extracting and identifying echinococcosis proteins and a limited number of echinococcosis antigens, this invention provides a polypeptide fragment (a neoantigen for human echinococcosis), a recombinant protein or its mutation, a nucleic acid sequence, and its application in the preparation of drugs for detecting or treating human echinococcosis. This invention expands the echinococcosis antigen database by identifying more antigens that can be used to diagnose echinococcosis, enabling these antigens, either individually or in combination, to potentially improve the specificity and sensitivity of laboratory diagnosis of echinococcosis.

[0007] A first aspect of the present invention provides a polypeptide comprising one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0008] A second aspect of the present invention provides a recombinant protein or a mutation thereof, wherein the recombinant protein comprises an amino acid sequence as shown in SEQ ID NO: 7; the mutated amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence of the recombinant protein, and maintains the function of the recombinant protein.

[0009] A third aspect of the present invention provides an isolated nucleic acid, wherein the isolated nucleic acid encodes the polypeptide described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention or a mutation thereof.

[0010] In some preferred embodiments, the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO:8.

[0011] A fourth aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid described in the third aspect of the present invention.

[0012] A fifth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the third aspect of the present invention or a recombinant expression vector as described in the fourth aspect of the present invention, wherein the transformant is a bacterium or a eukaryotic cell.

[0013] In some preferred embodiments, the bacteria is E. coli BL21.

[0014] In some preferred embodiments, the transformant comprises an amino acid sequence as shown in SEQ ID NO: 7.

[0015] The sixth aspect of the present invention provides a kit for detecting echinococcosis, wherein the kit comprises the polypeptide described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention or a mutation thereof.

[0016] In some preferred embodiments, the kit is an indirect ELISA detection kit.

[0017] In some preferred embodiments, the indirect ELISA detection kit further includes a second antibody, coating solution, washing solution, chromogenic solution, stop solution, and diluent.

[0018] A seventh aspect of the present invention provides a combination of biomarkers, wherein the combination of biomarkers includes the polypeptide described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention or a mutation thereof.

[0019] In some preferred embodiments, the biomarker combination further includes one or more of Egr, antigen B, and antigen 5.

[0020] The eighth aspect of the present invention provides an siRNA or mRNA vaccine against echinococcosis, wherein the siRNA or mRNA vaccine comprises an RNA sequence complementary to the nucleotide sequence encoding the polypeptide described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention or a mutated nucleotide sequence thereof.

[0021] The ninth aspect of the present invention provides a method for producing the polypeptide described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention or a mutation thereof, wherein the method comprises the following steps: culturing the transformant described in the fifth aspect of the present invention under conditions suitable for its fermentation, so as to express the polypeptide or recombinant protein or a mutation thereof.

[0022] The tenth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention, or the recombinant protein described in the second aspect of the present invention, or a combination of its mutations or the biomarkers described in the seventh aspect of the present invention, in the preparation of anti-echinococcosis antibodies or in diagnostic agents for diagnosing diseases caused by Echinococcus granulosus.

[0023] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0024] The reagents and raw materials used in this invention are all commercially available.

[0025] The positive and progressive effects of this invention are as follows:

[0026] (1) A new echinococcosis antigen SPON1-1 is provided, which can be used to make ELISA kits and for clinical detection of human echinococcosis; and can complement the Egr antigen to perform more sensitive detection of echinococcosis.

[0027] (2) The new echinococcosis antigen can be purified in large quantities, which facilitates large-scale screening of human echinococcosis. Attached Figure Description

[0028] Figure 1 The SDS-PAGE and Western blotting results of the whole proteins extracted from the five cyst fluid fractions are shown. The left image shows the WB results using serum from Patient No. 1 as the primary antibody and the five cyst fluid fractions as antigens. The middle image shows the WB results using serum from normal Tibetan individuals as the primary antibody and the five cyst fluid fractions as antigens. The right image shows the protein gelation results of the five cyst fluid fractions.

[0029] Figure 2 The purification results of SPON1-1 recombinant protein are shown in the figure. The figure shows the gradient elution results of SPON1-1 recombinant protein, with a sample volume of 10 μl in each lane. Each lane represents, in turn, the whole bacterial protein after sonication, the flow-through after loading onto the nickel column, and the elution gradient from 20 mM imidazole to 1 M imidazole.

[0030] Figure 3 This is a Western blot (WB) validation result of the purified SPON1-1 recombinant protein. Numbers 1-9 refer to serum samples from nine patients taken before hydatid cyst removal surgery. Control refers to serum from healthy, disease-free Tibetans. Egr_SDS-PAGE shows the protein gel electrophoresis results of the commercial antigen Egr; the protein band indicated by the arrow represents Antigen B.

[0031] Figure 4 This is a presentation of ELISA test results. SPON1-1_OD(+) and Egr_OD(+) refer to the OD values ​​of 607 ultrasound-positive plasma samples, while SPON1-1_OD(-) and Egr_OD(-) refer to the OD values ​​of 636 normal plasma samples.

[0032] Figure 5 The ROC curve results are shown below. The left graph shows the ROC curve for SPON1-1. Criterion refers to the value of SPON1-1 in serum testing: a value greater than 0.68 indicates a positive result (hydatid disease), and a value less than or equal to 0.68 indicates a negative result (no hydatid disease). The right graph shows the ROC curve for Egr. Criterion refers to the value of Egr in serum testing: a value greater than 0.99 indicates a positive result (hydatid disease), and a value less than or equal to 0.99 indicates a negative result (no hydatid disease). Detailed Implementation

[0033] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0034] Example 1: The process of identifying more hydatid proteins from hydatid cysts isolated from patients after surgery.

[0035] (1) First, the hydatid cysts isolated from 10 patients with hydatid disease after surgery were divided into four parts according to their tissue structure and proteins were extracted. From the inside out, they were the protocercariae, cyst fluid, germinal layer and keratinocyte. Then, according to whether they were in the active period, the 10 hydatid cysts were divided into an active hydatid cyst group and an inactive hydatid cyst group.

[0036] (2) Proteins were extracted from all four components of each hydatid cyst, and the extracted proteins were subjected to liquid enzymatic digestion and then identified by LC-MS / MS using a QE mass spectrometer.

[0037] (3) The raw data from the QE machine were used to identify echinococcosis protein using the maxquant protein identification software and the human and echinococcosis protein database. The identification results were statistically analyzed and the echinococcosis cysts with high echinococcosis protein content were identified. The results are shown in Table 1 and Table 2.

[0038] (4) As can be seen from Table 1 and Table 2, there are two basic conclusions: First, the tissue parts in which more hydatid protein was identified are the cyst fluid and the protocercariae; second, only the cyst fluid and protocercariae of the active hydatid cyst group contain more hydatid protein.

[0039] Table 1. Protein identification results of the active echinococcosis cysts

[0040] _1 represents the protocephala; _2 represents the cyst fluid; _3 represents the germinal layer; _4 represents the keratinocyte.

[0041] Table 2. Protein identification results of non-active echinococcosis cysts

[0042]

[0043] _1 represents the protocephala; _2 represents the cyst fluid; _3 represents the germinal layer; _4 represents the keratinocyte.

[0044] Example 2: Western Blot validation, gel digestion, and mass spectrometry identification (GeLC-MS / MS)

[0045] (1) Next, based on the QE identification results of the cyst fluid and protocercariae of 10 samples, we selected the protocercariae and cyst fluid components of five active echinococcosis cyst groups No.1-No.5 and performed WB experiments with patient serum to verify whether there was an immune response between the serum of echinococcosis patients and normal Tibetans and the extracted whole protein.

[0046] (2) Because the total amount of protein extracted from the protocercariae of some samples was very small, Western blotting experiments were mainly performed on the proteins of the cyst fluid components. The results are as follows: Figure 1 As shown, using the serum of patient No. 1 as an example, the immunoreaction patterns of serum from patients with echinococcosis and normal Tibetans with the five protein components of the cyst fluid are presented. The immunoreaction patterns of serum from other patients and normal Tibetans with the five sample proteins are similar to those of patient No. 1.

[0047] (3) Based on the SDS-PAGE patterns of these five cyst fluid components, we performed gel digestion on the SDS-PAGE gel, comparing it with the immunohistochemical bands obtained from Western blotting. This was done to isolate and identify more echinococcosis proteins in the corresponding molecular weight ranges, thereby increasing the probability of finding echinococcosis antigens that react with serum. In addition, this can reduce the impact of high-abundance proteins on identification.

[0048] (4) In order to reduce losses and reduce workload, we divided the five cyst fluid components into 10 components based on the WB immune bands, and then each component was subjected to intragel enzymatic hydrolysis.

[0049] (5) After LC-MS / MS analysis by the QE-HF-X mass spectrometer, the protein library was identified using the same database as in 2.2.1 using the maxquant software. The results are shown in Table 3.

[0050] (6) Data analysis shows that the echinococcosis proteins identified by gel separation of samples 1_2, 2_2 and 5_2 include the echinococcosis proteins identified in the other four samples. Therefore, the candidates for subsequent echinococcosis antigens should be found from the echinococcosis proteins identified in the components of these three samples.

[0051] Table 3. Protein identification results from enzymatic digestion of five capsule fluid components.

[0052]

[0053] The numbers in parentheses represent the total number of proteins identified, while the numbers outside the parentheses represent the echinococcosis proteins in each fraction. _2 represents cyst fluid.

[0054] Example 3: Screening for echinococcosis proteins as candidate antigens

[0055] (1) Common proteins identified in the same component in three samples were used as candidate antigen libraries, and a total of 93 unique proteins were generated from the 10 components.

[0056] (2) These 93 proteins were first compared with human protein databases for homology. After deleting proteins with particularly high homology and those that had been selected as echinococcosis antigens, the remaining proteins were used to predict B cell antigen epitopes.

[0057] Example 4: Antigenic epitope prediction and recombinant plasmid construction

[0058] (1) Antigen epitope prediction used an online prediction tool http: / / tools.iedb.org / bcell / . The protein sequence was imported and the Bepipred Linear Epitope Prediction 2.0 prediction method was selected to obtain a list of sequences that the protein may become an antigen epitope.

[0059] (2) Based on the list of antigenic epitopes for each candidate protein, we select sequence fragments containing one or more epitopes to form the sequence of the recombinant protein, and import this sequence into the alignment tool to compare it with the human protein database. Only sequence fragments with a similarity of <=20% to human proteins are selected to construct the recombinant protein.

[0060] (3) Finally, 14 protein sequence fragments were inserted into the pET-30a(+) plasmid to construct a recombinant plasmid using molecular cloning technology, and the insertion of the fragments was confirmed by sequencing.

[0061] Example 5: Expression and purification of recombinant proteins

[0062] (1) The recombinant plasmid was transferred into BL21(DE3) for small-scale expression. After confirming that the protein could be expressed and that the molecular weight was correct, it was then expanded.

[0063] (2) After the collected bacterial culture was subjected to ultrasonic protein extraction, it was then purified by nickel column chromatography using a His-tag carried by a plasmid. Figure 2 The purification results of the recombinant protein constructed from the SPON1-1 fragment of this invention are shown.

[0064] Example 6: Western Blotting Validation of SPON1-1 Recombinant Protein

[0065] (1) Eight of the 14 recombinant proteins were successfully expressed. Therefore, the purified eight recombinant proteins were subjected to Western blotting experiments with the serum of nine postoperative patients. The results showed that only four recombinant proteins had a large immune response to the patient serum, including the SPON1-1 recombinant protein. The SPON1-1 recombinant protein was subsequently used as a candidate echinococcosis antigen for ELISA verification experiments with more patient serum.

[0066] (2) Figure 1 Experiments have shown that the total protein in the cyst fluid has a strong immune response to the serum of patients, but has almost no response to the serum of normal Tibetans, and most of the protein bands that cause an immune response are not high-abundance protein bands. Figure 3 The Western blot (WB) results of SPON1-1 recombinant protein and commercial antigen Egr with serum from nine patients are presented. The immunoreactivity rate of SPON1-1 recombinant protein was 78%, and the immunoreactivity rate of commercial antigen was 67% (only samples with a clear band between 43KD and 34KD were counted). Table 4 shows the B-cell antigenic epitopes, nucleotide sequence, and amino acid sequence of this recombinant protein.

[0067] Table 4. B-cell antigenic epitopes, nucleotide sequences, and amino acid sequences of SPON1-1 recombinant protein.

[0068]

[0069] Example 7: ELISA assay of recombinant antigen SPON1-1 recombinant protein

[0070] The subjects tested were 607 ultrasound-positive plasma samples (including suspected cases) and 636 normal plasma samples (confirmed negative). The indirect ELISA was performed according to standard operating procedures. The results showed that the positive detection rate of SPON1-1 recombinant protein was 84%, and the negative detection rate was 89%. When the same plasma was tested with commercial antigen Egr (product name: purified Echinococcus granulosus antigen, catalog number: YM-VI08, supplier: Hangzhou Yiminuo Biotechnology Co., Ltd.), the positive detection rate was 86% and the negative detection rate was 92%.

[0071] 1. Detailed operating procedures for ELISA experiments

[0072] (1) Antigen quantification: Each antigen was mixed with a pipette before coating and quantified using a micro-ultraviolet spectrophotometer to ensure that the protein was not degraded. If there was no absorption peak at A280, it indicated that the amount of antigen was too low to be used for coating; if the protein precipitated or was insoluble, 8M urea was added and mixed, and the supernatant was taken by centrifugation to measure the concentration.

[0073] (2) Coating: Compare the antigen concentration on the tube wall with the measured value and take the lower value; the coating amount is 2 μg / ml or 10 ml / plate, and the amount of coating antigen is prepared according to the actual usage; the coated ELISA plate is labeled with: antigen number, name, label, coating date and plate number, etc. If the coating concentration is not 2 μg / ml, the coating concentration needs to be indicated. After labeling the ELISA plate, add the coating antigen, 100 μl / well, and coat overnight at 4℃ or for 2 hours at 37℃.

[0074] (3) Plate washing: After coating, wash the plates once with a plate washer and pat dry on absorbent paper. The 50X washing solution formula is: Tris 154.4 g, NaCl 149.0 g, Tween 2024.0 ml, pure water 800 ml, adjust the pH to 7.2 with about 45 ml of concentrated hydrochloric acid, and bring the volume to 1000 ml with pure water. Store at 4℃.

[0075] (4) Blocking: 2% skim milk powder is used as the blocking solution for blocking, 200 μl / well, and the solution is blocked overnight at 4°C or blocked for 2 hours at 37°C.

[0076] (5) Washing the board: After sealing, wash the board once with a board washing machine and pat it dry on absorbent paper.

[0077] (6) Add primary antibody: Add the corresponding primary antibody, dilute the plasma with PBS at a ratio of 1:500, and incubate at 37°C for 1 hour.

[0078] (7) Washing the plate: Wash the plate more than 3 times with a plate washing machine and pat it dry on absorbent paper.

[0079] (8) Add secondary antibody (manufacturer: Beyotime; product number: A0201): Since the primary antibody is derived from human plasma, the secondary antibody is added at a ratio of 1:500 using goat anti-human and incubated at 37°C for 1 hour.

[0080] (9) Plate washing: Wash the plate at least 3 times with a plate washer and pat it dry on absorbent paper. While washing the plate, prepare the color development solution (TMB).

[0081] (10) Color development: After preparing the stop solution, add 100 μl of color development solution per well. You can shake the plate to accelerate the color development process and pay close attention to observation.

[0082] (11) TMB color development: When the color development reaches a certain level (generally, the blank and negative color development should not be too high), add 50 μl / well of stop solution. After adding the stop solution, it needs to stand for more than 10 minutes to ensure complete termination and uniform color (you can shake the plate to speed up the termination process). Read the value after termination.

[0083] (12) Reading: The microplate reader must be preheated for at least 30 minutes; the TMB colorimetric detection wavelength is 450 nm. Open the corresponding microplate reader measurement software and read the data. Store the data in the designated location and complete the data.

[0084] (13) Precautions:

[0085] When adding samples to a 96-well plate using a pipette, be careful to avoid generating air bubbles and to prevent the added sample from sticking to the plate.

[0086] After adding the sample, observe the entire plate, ensuring there are no air bubbles at the bottom of the wells; if air bubbles are found, shake the plate or remove them with a pipette tip.

[0087] Close attention must be paid during the cleaning process to ensure that the cleaning is complete and thorough.

[0088] Close monitoring is required during color development, and the process should be terminated promptly based on the color development results of negative, blank, and positive controls.

[0089] (14) Formulas for various reagents

[0090] Coating solution: Sodium carbonate-sodium bicarbonate buffer, pH 9.6: Na₂CO₃ 1.59 g, NaHCO₃ 2.93 g, diluted to 1000 ml with pure water; finally, check the pH value using pH test paper. Store at 4℃.

[0091] 2. ELISA Result Analysis

[0092] (1) Criteria for judging positive and negative results: The mean (X) and standard deviation (SD) of negative serum samples are selected, and the upper limit of the confidence interval is X+2SD. The OD value of the test sample at 450nm is greater than or equal to X+2SD and is judged as positive, and less than X+2SD and is judged as negative.

[0093] (2) ROC curve: The ROC curve (Receiver Operating Characteristic curve) was plotted using MedCalc software (version 19.4.0) to evaluate the optimal detection power of the antigen.

[0094] (3) By calculation, the sensitivity of immunizing 607 cases of ultrasound-positive plasma (including suspected cases) and 636 cases of normal plasma (confirmed negative) with the echinococcosis antigen of the present invention was 84% ​​and the specificity was 89%; while the sensitivity of detecting the same plasma with commercial antigen Egr (hereinafter referred to as commercial antigen or Egr) was 86% and the specificity was 92%. Figure 4 The ROC curves of SPON1-1 recombinant protein and Egr, as well as box plots for detecting OD values, are shown.

[0095] (4) Although the specificity of the SPON1-1 recombinant protein is slightly lower than that of the commercial antigen, its sensitivity is basically the same. Furthermore, analysis using MedCalc software yielded dual-characteristic ROC curves for both the SPON1-1 recombinant antigen and Egr, showing that the combined detection efficacy of the two antigens is excellent. This indicates that the SPON1-1 recombinant antigen could potentially be used as a supplementary detection method to retest samples that were not detected positive by the commercial antigen. Therefore, the SPON1-1 recombinant protein can be considered a candidate for a new echinococcosis antigen. SEQUENCE LISTING <110> Shenzhen BGI Life Science Research Institute <120> A SPON1-1 protein fragment and its application in human echinococcosis <130> P22012951C <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Table 1 <400> 1 Trp Arg Ser Pro Asn Ala Ser Arg His Asn Cys Val Thr Leu His Ala 1 5 10 15 Met Val Arg Thr Arg Thr Ser Val Leu Lys Glu Val Gly Gly Leu 20 25 30 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Epitope 2 <400> 2 Lys Thr Leu Cys Pro Thr Glu Met Arg Pro Met Glu Pro 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Epitope 3 <400> 3 Ser Arg Ile Arg Glu Ser Leu Asp Val 1 5 <210> 4 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Epitope 4 <400> 4 Met Gln Thr Thr Leu Arg Asp Ser Val Gln Pro Lys Asp Cys Cys Ser 1 5 10 15 Cys Gly Thr Ala Thr Tyr Arg Leu Thr Phe Gln Gly Leu Trp Asn Arg 20 25 30 Ser Thr His Pro Gln Asp Trp Pro Thr Lys Asn Pro Asn Leu Leu His 35 40 45 Trp Thr Asn Leu Ile Gly Ala Ser His Ala Pro Ser Phe Gln Ile Tyr 50 55 60 Ala Ile Gly Gln Thr Ala Ser Ala Gly Val Gln Ser Val Cys Ala Tyr 65 70 75 80 Gly Asp Thr Thr Val Leu Arg Glu Ala Leu Ser Leu Ala Ala Ala Lys 85 90 95 Ala Glu Gly Ala Ala Gly Val Pro Thr Gly Ser Gln Ser Ala Thr Thr 100 105 110 Asp Ile Ser Pro 115 <210> 5 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Epitope 5 <400> 5 Trp Gly Glu Glu Thr Leu Met Glu Arg Arg Thr Thr Leu Phe Ser Val 1 5 10 15 Asn Arg Thr His Pro Leu Phe Ser Met Leu Thr Met Leu Gly Pro Ser 20 25 30 Pro Asp Trp Cys Thr Gly Ile Ser Gly Gln Ser Leu Cys Lys Ala Asp 35 40 45 Cys Thr Trp Val Arg Asn Met Thr Leu Tyr Leu His 50 55 60 <210> 6 <211> 12 <212> PRT​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Val Gln Pro Lys Asp Cys Cys Ser Cys Gly Thr Ala Thr Tyr Arg Leu 85 90 95 Thr Phe Gln Gly Leu Trp Asn Arg Ser Thr His Pro Gln Asp Trp Pro 100 105 110 Thr Lys Asn Pro Asn Leu Leu His Trp Thr Asn Leu Ile Gly Ala Ser 115 120 125 His Ala Pro Ser Phe Gln Ile Tyr Ala Ile Gly Gln Thr Ala Ser Ala 130 135 140 Gly Val Gln Ser Val Cys Ala Tyr Gly Asp Thr Thr Val Leu Arg Glu 145 150 155 160 Ala Leu Ser Leu Ala Ala Ala Lys Ala Glu Gly Ala Ala Gly Val Pro 165 170 175 Thr Gly Ser Gln Ser Ala Thr Thr Asp Ile Ser Pro Leu Arg Ala Leu 180 185 190 Ile Phe Thr Pro Gly Met Trp Gly Glu Glu Thr Leu Met Glu Arg Arg 195 200 205 Thr Thr Leu Phe Ser Val Asn Arg Thr His Pro Leu Phe Ser Met Leu 210 215 220 Thr Met Leu Gly Pro Ser Pro Asp Trp Cys Thr Gly Ile Ser Gly Gln 225 230 235 240 Ser Leu Cys Lys Ala Asp Cys Thr Trp Val Arg Asn Met Thr Leu Tyr 245 250 255 Leu His Pro Trp Asp Ala Gly Ile Arg Glu Gly Asn Thr Tyr Met Pro 260 265 270 Lys Glu Ser Asp Arg 275 <210> 8 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> spon1‐1 <400> 8 tggcgtagtc caaatgccag ccgccacaac tgcgttacac ttcatgccat ggtgcgtaca 60 cgcacatcag ttctgaagga agttggtggt ttgatgaaga cactttgccc cactgaaatg 120 cgtccgatgg agccagtgat ctcgagaata cgtgaaagtc ttgacgttga ccgaaatgat 180 cctacagttt acctccaacc accctacgca aatcctatgc aaacgacact tcgagacagc 240 gttcaaccaa aggattgttg ttcatgcggt accgccacct acagactgac tttccaaggt 300 ctctggaacc gatcgactca ccctcaagac tggcccacca aaaatccaaa tctccttcac 360 tggacgaacc tcatcggcgc cagtcatgca cccagcttc agatttatgc catcggacaa 420 acggctagtg caggagtcca gtctgtctgt gcatatggag acactacggt gctccgtgaa 480 gccctctcct tggctgctgc aaaggctgaa ggagctgcgg gagttccaac tggtagtcaa 540 tcggctacaa ccgacatcag cccccttcga gcacttattt tcacccctgg tatgtggggt 600 gaggagacgc tgatggagcg gcgtacaacg cttttctcag taaaccgaac ccatccactc 660 ttttcgatgc ttacaatgct gggtccgagt cctgactggt gtacgggcat ttccggccag 720 tcattatgca aggcggactg tacgtgggtg aggaatatga cattatattt gcacccctgg 780 gatgcaggaa ttagagaagg aaatacgtat atgccaaagg aatcggatag gtaa 834

Claims

1. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown as SEQ ID NO:

7.

2. An isolated nucleic acid, comprising, The isolated nucleic acid encodes the recombinant protein of claim 1.

3. The isolated nucleic acid of claim 2, wherein, The nucleotide sequence of the isolated nucleic acid encoding the recombinant protein of claim 1 is shown as SEQ ID NO:

8.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid of claim 2 or 3.

5. A transformant characterized in that, It comprises the isolated nucleic acid of claim 2 or 3 or the recombinant expression vector of claim 4, wherein the transformant is a bacterial or eukaryotic cell.

6. The transformant of claim 5, wherein, The bacteria is E. coli BL21.

7. The transformant according to claim 5 or 6, wherein The transformant expresses the recombinant protein with the amino acid sequence shown as SEQ ID NO:

7.

8. A kit for detecting echinococcosis, characterized by, The kit comprises the recombinant protein of claim 1.

9. The kit of claim 8, wherein The kit is an indirect ELISA detection kit.

10. The kit of claim 9, wherein The indirect ELISA detection kit further comprises a secondary antibody, a coating solution, a washing solution, a color developing solution, a termination solution and a dilution solution.

11. A marker combination, characterized in that The marker combination comprises the recombinant protein of claim 1, wherein the marker combination is a marker combination for echinococcosis.

12. The marker combination of claim 11, wherein, The marker combination further comprises one or more of Egr, antigen B and antigen 5.

13. A method of producing the recombinant protein of claim 1, wherein, The method comprises the following steps: culturing the transformant of any one of claims 5-7 under conditions suitable for its fermentation to express the recombinant protein.

14. Use of the recombinant protein of claim 1 or the marker combination of claim 11 or 12 in the preparation of an antibody against echinococcosis or in the preparation of a diagnostic agent for diagnosing diseases caused by Echinococcus granulosus.

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