A UP protein fragment and application thereof in human hydatidosis
By providing a novel human echinococcosis antigen, the UP protein fragment, and its nucleic acid sequence, combined with the existing antigen Egr, the problems of limited antigen types and false positives/false negatives in echinococcosis diagnosis have been solved, achieving more accurate detection and treatment results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN HUADA GENE INST
- Filing Date
- 2022-06-08
- Publication Date
- 2026-04-10
AI Technical Summary
The limited number of diagnostic antigens for echinococcosis in existing technologies makes it easy to produce false positives or false negatives in serum testing, and makes the extraction and identification of echinococcosis proteins difficult.
This invention provides a polypeptide, namely a fragment of the novel human echinococcosis antigen UP protein and its nucleic acid sequence, which is expressed in bacteria or eukaryotic cells via a recombinant expression vector for the preparation of kits and vaccines for the detection or treatment of human echinococcosis, and can be detected in combination with the existing antigen Egr.
It improves the specificity and sensitivity of echinococcosis detection, reduces false positive and false negative results, and provides a more accurate diagnostic tool.
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Figure CN115850424B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of immunology, in particular to a polypeptide, i.e. a novel antigen UP protein fragment of human echinococcosis, a nucleic acid sequence and application thereof in preparation of a drug for detecting or treating human echinococcosis. BACKGROUND
[0002] Echinococcosis, also known as hydatid disease, is a zoonosis. Human echinococcosis is mainly divided into cystic echinococcosis and alveolar echinococcosis. Imaging detection is the most intuitive and most commonly used method for diagnosing echinococcosis, but because the incubation period of echinococcosis is long, and only when the cyst formed by echinococcosis reaches a certain size can it be detected by imaging, serological detection is one of the important auxiliary means for imaging diagnosis of echinococcosis. At present, the serological diagnostic antigens that can be used to diagnose echinococcosis mainly include Antigen B (Antigen B) and Antigen 5 (Antigen 5) and some recombinant antigens related to the two antigens.
[0003] It is a technical difficulty to extract and identify echinococcal proteins from the cysts of postoperative echinococcosis patients. It will be more difficult to select proteins that may serve as echinococcal antigens from the few echinococcal proteins identified.
[0004] Secondly, the single antigen is few. The most widely used echinococcal diagnostic antigen is Antigen B and Antigen 5, and many studies have shown that these two antigens will produce certain false positives or false negatives in serum detection.
[0005] Commercial echinococcal antigens are natural purified antigens, which are isolated and purified from soluble antigen fragments after crushing of fine-grained echinococcosis. It is a mixture containing multiple echinococcal proteins. The more mixed proteins, the more likely it is to produce more false negatives or false positives in serum detection. SUMMARY
[0006] To solve the technical defects of echinococcal protein extraction and identification difficulty and few echinococcal antigens in the prior art, the present application provides a polypeptide, i.e. a novel antigen UP protein fragment of human echinococcosis, a nucleic acid sequence and application thereof in preparation of a drug for detecting or treating human echinococcosis. The present application finds more antigens that can be used to diagnose echinococcosis, expands the echinococcal antigen database, and makes it possible to improve the specificity and sensitivity of laboratory diagnosis of echinococcosis by these antigens alone or in combination.
[0007] The first aspect of the present application provides a polypeptide, wherein the polypeptide comprises one or more of the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
[0008] The second aspect of the present application provides an isolated nucleic acid, wherein the isolated nucleic acid encodes the polypeptide according to the first aspect of the present application.
[0009] The third aspect of the present application provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid according to the second aspect of the present application.
[0010] The fourth aspect of the present application provides a transformant comprising the isolated nucleic acid according to the second aspect of the present application or the recombinant expression vector according to the third aspect of the present application, wherein the transformant is a bacterial or eukaryotic cell.
[0011] In some preferred embodiments, the bacteria is E. coli BL21.
[0012] The fifth aspect of the present application provides a kit for detecting echinococcosis, wherein the kit comprises the polypeptide according to the first aspect of the present application.
[0013] In some preferred embodiments, the kit is an indirect ELISA detection kit.
[0014] In some more preferred embodiments, 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.
[0015] The sixth aspect of the present application provides a marker combination, wherein the marker combination comprises the polypeptide according to the first aspect of the present application.
[0016] In some preferred embodiments, the marker combination further comprises one or more of Egr, antigen B and antigen 5.
[0017] The seventh aspect of the present application provides an anti-echinococcosis siRNA or mRNA vaccine, wherein the siRNA or mRNA vaccine comprises an RNA sequence complementary to a nucleotide sequence encoding the polypeptide according to the first aspect of the present application.
[0018] The eighth aspect of the present application provides a method for producing the polypeptide according to the first aspect of the present application, wherein the method comprises the following steps: culturing the transformant according to the fourth aspect of the present application under conditions suitable for its fermentation, so that it expresses the polypeptide.
[0019] The ninth aspect of the present application provides a polypeptide according to the first aspect of the present application or a marker combination according to the sixth aspect of the present application for use in the preparation of an antibody against echinococcosis or a diagnostic agent for diagnosing a disease caused by echinococcus granulosus.
[0020] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred embodiments of the present application.
[0021] The reagents and raw materials used in the present application are commercially available.
[0022] The positive progress effect of the present application is that:
[0023] (1) A new antigen UP of echinococcus is provided, which can be used to prepare an ELISA kit and for clinical detection of human echinococcosis; and can be used as a supplement to Egr antigen for more sensitive detection of echinococcosis.
[0024] (2) The new antigen of echinococcus can be purified in large quantities, facilitating large-scale screening of human echinococcosis. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 SDS-PAGE and Western blotting results of total proteins extracted from five cyst fluid components are shown. The left panel shows the WB results of using No. 1 patient serum as primary antibody and five cyst fluid components as antigen. The middle panel shows the WB results of using normal Tibetan serum as primary antibody and five cyst fluid components as antigen. The right panel shows the protein gel results of five cyst fluid components.
[0026] Figure 2 The purification results of UP recombinant protein are shown. The gradient elution results of UP recombinant protein are shown in the figure, and the loading amount of each lane is 10 μl. Each lane is sequentially labeled as total protein after ultrasonic treatment, flow-through after loading on a nickel column, and elution gradient from 20 mM imidazole to 1 M imidazole.
[0027] Figure 3 The WB verification experiment results of UP recombinant protein after purification are shown. Numbers 1-9 refer to 9 patient sera taken before echinococcal cyst removal surgery. Control refers to normal Tibetan serum without disease. Egr_SDS-PAGE refers to the protein gel results of commercial antigen Egr, and the protein band indicated by the arrow is Antigen B.
[0028] Figure 4 The ELISA experiment results are shown. UP_OD(+) and Egr_OD(+) refer to the OD values of 607 cases of B-ultrasound positive plasma, and UP_OD(-) and Egr_OD(-) refer to the OD values of 636 cases of normal human plasma.
[0029] Figure 5 ROC curve of UP recombination protein and commercial antigen Egr. The left graph is the ROC curve of UP, and Criterion refers to the detection of UP on serum, greater than 0.6 is positive (suffering from echinococcosis), and less than or equal to 0.6 is negative (not suffering from echinococcosis). The right graph is the ROC curve of Egr, and Criterion refers to the detection of Egr on serum, greater than 0.99 is positive (suffering from echinococcosis), and less than or equal to 0.99 is negative (not suffering from echinococcosis). DETAILED DESCRIPTION
[0030] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples are not specified, and the methods are selected according to the conventional methods and conditions, or according to the product instructions.
[0031] Example 1: Process of identifying more echinococcus proteins from the cysts of echinococcosis patients after operation
[0032] (1) First, 10 cysts of echinococcosis patients after operation were divided into four parts according to their tissue structure, and proteins were extracted from the inside out, i.e., the protoscolex, the cyst fluid, the germinal layer and the cuticula layer; and according to whether they were in the active stage, the 10 cysts were divided into the active cyst group and the non-active cyst group.
[0033] (2) Protein extraction was performed on each of the four components of the cyst, and the extracted protein was subjected to liquid enzymatic hydrolysis, followed by LC-MS / MS identification using QE mass spectrometer;
[0034] (3) The raw data from QE was subjected to protein identification using maxquant protein identification software, using a combined database of human and echinococcus proteins, and the identification results were counted and the parts of the cyst with more echinococcus protein content were determined, as shown in Tables 1 and 2;
[0035] (4) From Tables 1 and 2, two basic conclusions can be drawn: first, the parts with more echinococcus proteins identified are the cyst fluid and the protoscolex; second, only the cyst fluid and the protoscolex parts of the active cyst group contain more echinococcus proteins.
[0036] Table 1 Protein identification results of the active cyst group
[0037]
[0038]
[0039] _1 represents the protoscolex; _2 represents the cyst fluid; _3 represents the germinal layer; and _4 represents the cuticula layer.
[0040] Table 2 Protein identification results of non-active cyst group
[0041]
[0042] _1 indicates protoscolex; _2 indicates cyst fluid; _3 indicates germinal layer; _4 indicates cuticula.
[0043] Example 2: Western Blot verification, in-gel digestion and mass spectrometry identification (GeLC-MS / MS)
[0044] (1) Next, according to the QE identification results of the cyst fluid and protoscolex of the 10 samples, we selected the protoscolex and cyst fluid components of the five active cyst groups No. 1-No. 5 to perform WB experiments with patient serum in order to verify whether there is an immune response between the serum of echinococcosis patients and normal Tibetans and the extracted whole protein.
[0045] (2) Because the total amount of protein extracted from the protoscolex component of some samples is very small, we mainly performed Western Blotting experiments on the cyst fluid component protein, and the results are shown in Figure 1 As an example, the immune response map of the serum of echinococcosis patients and normal Tibetans to the five cyst fluid component proteins is shown. The immune response maps of other patient serum and normal Tibetan serum to the proteins of the five samples are similar to those of No. 1 patient.
[0046] (3) According to the SDS-PAGE map of the five cyst fluid components, we performed in-gel digestion on the SDS-PAGE gel by comparing the WB immune bands in order to identify more echinococcosis proteins in the corresponding molecular weight segments through in-gel separation, thereby increasing the probability of finding echinococcosis antigens that have an immune response with serum. In addition, this can reduce the impact of high-abundance proteins on identification.
[0047] (4) In order to reduce losses and workload, we uniformly divided the five cyst fluid component proteins into 10 components according to the WB immune bands, and then each component was subjected to in-gel digestion.
[0048] (5) After LC-MS / MS analysis by QE-HF-X mass spectrometer, the maxquant software used the same database as in 2.2.1 for protein library identification. The results are shown in Table 3.
[0049] (6) Through data analysis, it can be known that the echinococcosis proteins identified by in-gel separation of the three samples 1_2, 2_2 and 5_2 contain the echinococcosis proteins identified by the other four samples, so the subsequent candidate of echinococcosis antigens is searched from the echinococcosis proteins identified from each component of the three samples.
[0050] Table 3 Protein identification results of five cystic fluid component gelatinase digestion
[0051]
[0052] The number in the bracket represents the total identified proteins, and the number outside the bracket represents the hydatid proteins in each component. _2 represents cystic fluid.
[0053] Example 3: Screening of hydatid proteins as candidate antigens
[0054] (1) The common proteins identified in the same component in three samples were used as a candidate antigen library, and finally 10 components produced a total of 93 unique proteins.
[0055] (2) The 93 proteins were first subjected to homology comparison with the human protein database, and after deleting proteins with high homology and those already selected as hydatid antigens, the remaining proteins were subjected to B cell antigen epitope prediction.
[0056] Example 4: Antigen epitope prediction and construction of recombinant plasmid
[0057] (1) The 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, i.e. the sequence list about the possible antigen epitope of the protein was obtained.
[0058] (2) We selected the sequence fragments containing one or more epitopes according to the antigen epitope list of each candidate protein to form the sequence of the recombinant protein, and imported the sequence into the alignment tool for comparison with the human protein database. Only the sequence fragments with a similarity of <= 20% to human proteins were selected to construct the recombinant protein.
[0059] (3) Finally, 14 protein sequence fragments were inserted into pET-30a(+) plasmid by molecular cloning technology to construct recombinant plasmid, and the fragments were confirmed to have been inserted by sequencing.
[0060] Example 5: Expression and purification of recombinant protein
[0061] (1) The recombinant plasmid was transformed into BL21(DE3) for small-scale expression, and after confirming that the protein could be expressed and the molecular weight was correct, it was expanded.
[0062] (2) The collected bacterial solution was subjected to ultrasonic extraction of protein, and then subjected to nickel column purification by His-tag carried by the plasmid, Figure 2 The purification results of the recombinant protein constructed by the UP fragment of the application are shown.
[0063] Example 6: Western Blotting verification of UP recombinant protein
[0064] (1) Among the 14 recombinant proteins, 8 were successfully expressed, so the 8 purified recombinant proteins were respectively subjected to Western Blotting experiment with 9 postoperative patient sera, and the results showed that only 4 recombinant proteins had larger immune reaction with patient sera, among which the UP recombinant protein was contained, and the UP recombinant protein was subsequently used as a candidate of hydatid antigen for more patient serum ELISA verification test.
[0065] (2) Figure 1 Experiments proved that the whole protein of cyst fluid had strong immune reaction with patient serum, and had no reaction with normal Tibetan serum, and most of the protein bands causing immune reaction were not high-abundance protein bands; Figure 3 The WB experiment results of UP recombinant protein and commercial antigen Egr with 9 patient sera were shown, the immune reaction rate of UP recombinant protein was 56%, and the immune reaction rate of commercial antigen was 67%(only samples with obvious bands between 43KD and 34KD were counted). 4 The B cell antigen epitope, nucleotide sequence and amino acid sequence of this recombinant protein were shown.
[0066] Table 4 B cell antigen epitope, nucleotide sequence and amino acid sequence of UP recombinant protein
[0067]
[0068]
[0069] Example 7: ELISA experiment of recombinant antigen UP recombinant protein
[0070] The detected objects were 607 cases of B-ultrasound positive plasma(including suspected) and 636 cases of normal human plasma(definite negative), and the standard operation procedure of indirect ELISA was performed, and the results showed that the positive detection rate of UP recombinant protein was 90%, and the negative detection rate was 77%; while the same plasma was detected by commercial antigen Egr(product name: hydatid purified antigen Echinococcus granulosus, product number: YM-VI08, supplier: Hangzhou Yiminuo Biological Technology Co., Ltd.), the positive detection rate was 86%, and the negative detection rate was 92%.
[0071] 1. Specific operation steps of ELISA experiment
[0072] (1) Antigen quantification: Before coating, each antigen was blown evenly with a pipette, and the antigen was quantified using a micro-UV spectrophotometer to ensure that the protein was not degraded. No absorption peak at A280 indicates that the antigen amount is too low to be used for coating; if the protein precipitates or is insoluble, add 8M urea and blow evenly, then centrifuge to obtain the supernatant for concentration measurement.
[0073] (2) Coating: For the wall concentration and measurement of the contrast antigen, take the lower value; the coating amount is 2 μg / ml, 10 ml / plate, and the coating antigen amount is prepared according to the actual use amount; the coated ELISA plate is labeled with antigen number, name, label, coating date, and plate number, etc., and 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°C or for 2 h at 37°C.
[0074] (3) Washing plate: After coating, the plate is washed once with a plate washer and dried on absorbent paper. The 50X washing solution is prepared as follows: Tris 154.4 g, NaCl 149.0 g, Tween 20 24.0 ml, pure water 800 ml, about 45 ml of concentrated hydrochloric acid to adjust pH 7.2, and pure water to 1000 ml. Store at 4°C.
[0075] (4) Blocking: 2% skimmed milk powder is used as blocking solution for blocking, 200 μl / well, 4°C blocking overnight or 37°C blocking for 2 h.
[0076] (5) Washing plate: After blocking, the plate is washed once with a plate washer and dried 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 h.
[0078] (7) Wash plate: Wash the plate with a plate washer for more than 3 times, and dry on absorbent paper.
[0079] (8) Add secondary antibody (manufacturer: Biyun Tian; item number: A0201): Since the primary antibody is derived from human plasma, goat anti-human 1:500 is used as the secondary antibody, and incubated at 37°C for 1 h.
[0080] (9) Wash plate: Wash the plate with a plate washer for more than 3 times, and dry on absorbent paper. At the same time, prepare the color developing solution (TMB).
[0081] (10) Color development: After preparing the stop solution, add 100 μl / well of color developing solution, shake the plate to accelerate the color development process, and observe closely.
[0082] (11) TMB color development: when the color development reaches a certain degree (generally, the blank and negative color development cannot be too high), 50 μl / well of stop solution is added, and after the addition of the stop solution, it needs to be rested for more than 10 min to make the termination thorough and the color uniform (the plate can be shaken to accelerate the termination process), and after termination, reading
[0083] (12) Reading: the enzyme label instrument must be preheated for more than 30 min; the TMB color development detection wavelength is 450 nm. Open the corresponding enzyme label instrument measurement software to read. Store the data to the designated position and perfect the data.
[0084] (13) Matters needing attention:
[0085] When adding samples to the 96-well plate with a gun, attention should be paid to avoid bubbles and to avoid the hanging of the added sample on the wall.
[0086] After adding the sample, the whole plate is observed, and it is required that there is no bubble at the bottom of the hole; when bubbles are found, the plate is shaken or the bubbles are removed with a gun head.
[0087] When washing the plate, close attention should be paid to ensure that the plate is completely and thoroughly washed.
[0088] When color development, close attention should be paid, combined with the color development of negative, blank and positive controls, and terminated in time.
[0089] (14) Various reagent formulations
[0090] Coating solution: sodium carbonate-sodium bicarbonate buffer, pH 9.6: Na2CO3 1.59 g, NaHCO3 2.93 g, and pure water to 1000 ml; finally, the pH value is detected with pH paper. Store at 4°C.
[0091] 2. ELISA result analysis
[0092] (1) Negative and positive judgment standard: the average value (X) of the negative serum sample is selected, the standard deviation (SD) is selected, and the upper limit of the confidence interval cut-off value is X+2SD. The OD value of the sample to be tested at 450 nm is greater than or equal to X+2SD, which can be judged as positive, and less than X+2SD, which can be judged as negative.
[0093] (2) ROC curve: MedCalc software (version 19.4.0) is used to draw the ROC curve (Receiver Operating Characteristic curve) to evaluate the optimal detection efficiency of the antigen.
[0094] (3) Through calculation, 607 cases of B-ultrasound positive plasma (including suspected) and 636 cases of normal human plasma (determined to be negative) are detected by using the hydatid antigen of the application, the sensitivity is 90%, and the specificity is 77%; and when the same plasma is detected by using the commercial antigen, the sensitivity is 86%, and the specificity is 92%.Figure 4 The ROC curve of UP recombinant protein and commercial antigen Egr and the box plot of detection OD value are shown.
[0095] (4) Although the specificity of UP recombinant protein is lower than that of commercial antigen, the sensitivity of UP recombinant protein is higher than that of commercial antigen, and the double feature ROC curve of UP recombinant antigen and commercial antigen Egr simulated by MedCalc software shows that the detection efficiency of the two antigens together is very good. It is shown that UP recombinant antigen can be used as a supplement to commercial antigen to retest the samples that are not detected positive by commercial antigen, so UP recombinant protein can be used as a candidate for new antigen of hydatid disease. SEQUENCE LISTING <110> Shenzhen Huada Kangle Gene Research Institute <120> UP protein fragment and its application in human hydatid disease <130> P22012953C <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Epitope 1 <400> 1 Lys Pro Gln Lys Ile Thr Val Asp Thr Lys Ser Cys Gly Pro Leu Thr 1 5 10 15 Ala Tyr Leu His Gly Gly Arg Val Gly Lys Asp Val Ala Ala Leu Thr 20 25 30 Val His Asp Leu Gly 35 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Epitope 2 <400> 2 Phe Val Asn Asn Glu His Met Ser Gin Leu Thr Gin Arg 1 5 10 <210> 3 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Epitope 3 <400> 3 Val Pro Gly Gin Gly Asp Asn Met Pro Asp Leu Pro Ser Asp Trp Lys 1 5 10 15 Phe Pro Thr Met Gin Lys He Ser Glu Gly He Ser Glu Leu Cys Asp 20 25 30 Ser Leu Glu Leu Lys His Val Val Val He Gly Asp Gly Ala Gly Ala 35 40 45 Asn He Val Ala Arg Leu Ala Met Ala Arg Glu Asp 50 55 60 <210> 4 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Epitope 4 <400> 4 Leu He His Cys Thr Gly Thr Thr Ala Gly Phe Met Glu Ser Leu Arg 1 5 10 15 Asp Arg Val Asn Ser Trp Lys Leu Asn Thr He Gly Met His Pro Ser 20 25 30 Val <210> 5 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Epitope 5 <400> 5 Gln Ser Leu Arg Glu Asn Ile Asn Pro Lys Asn Leu Asn Lys Phe Ile 1 5 10 15 Gln Ala Phe Met Val Arg Thr Asn Ile Thr Asp Ser Ile Gly Asn Leu 20 25 30 Lys Cys <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Epitope 6 <400> 6 Ile Thr Gly Ser Val Ala Ser Phe Asn His Thr Val Tyr Thr Leu Tyr 1 5 10 15 Asn Ala <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Epitope 7 <400> 7 Pro Ala Arg Lys Ala Asn Val Glu Ile Leu Glu Ile Asp 1 5 10 <210> 8 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Epitope 8 <400> 8 Glu Lys Val Ala Glu Ser Val Gin Asn Phe Met Gin Gly Leu Gly Val 1 5 10 15 Ala Ser Gly Ala Val Asn Arg Arg Leu Ser Ala Thr Gly Asn Val Pro 20 25 30 Lys lie Arg Asn Arg Ser Ala Ser Met Asp Glu Tyr Asp Gin 35 40 45 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Epitope 9 <400> 9 Ile Tyr Asp Asn Thr Arg Arg Tyr Ser Lys Ala Thr Asp lie Pro Glu 1 5 10 15 Ile Ser Glu Val 20 <210> 10 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> UP <400> 10 Met Ser Ser Gin Lys Pro Gin Lys lie Thr Val Asp Thr Lys Ser Cys 1 5 10 15 Gly Pro Leu Thr Ala Tyr Leu His Gly Gly Arg Val Gly Lys Asp Val 20 25 30 Ala Ala Leu Thr Val His Asp Leu Gly Tyr Asn Glu Phe Phe Glu Phe 35 40 45 Val Asn Asn Glu His Met Ser Gln Leu Thr Gln Arg Val Phe Trp Ile 50 55 60 His Val Glu Val Pro Gly Gln Gly Asp Asn Met Pro Asp Leu Pro Ser 65 70 75 80 Asp Trp Lys Phe Pro Thr Met Gln Lys Ile Ser Glu Gly Ile Ser Glu 85 90 95 Leu Cys Asp Ser Leu Glu Leu Lys His Val Val Val Ile Gly Asp Gly 100 105 110 Ala Gly Ala Asn Ile Val Ala Arg Leu Ala Met Ala Arg Glu Asp Ile 115 120 125 Cys Leu Gly Ala Ile Leu Ile His Cys Thr Gly Thr Thr Ala Gly Phe 130 135 140 Met Glu Ser Leu Arg Asp Arg Val Asn Ser Trp Lys Leu Asn Thr Ile 145 150 155 160 Gly Met His Pro Ser Val Glu Asn Tyr Leu Val Leu His Arg Phe Gly 165 170 175 Val Phe Ile Lys Ala Thr Thr Glu Ala Glu Leu Arg Gly Ala Ile Lys 180 185 190 Asn Phe Leu Gin Ser Leu Arg Glu Asn lie Asn Pro Lys Asn Leu Asn 195 200 205 Lys Phe lie Gin Ala Phe Met Val Arg Thr Asn lie Thr Asp Ser lie 210 215 220 Gly Asn Leu Lys Cys Pro Val Leu Phe lie Thr Gly Ser Val Ala Ser 225 230 235 240 Phe Asn His Thr Val Tyr Thr Leu Tyr Asn Ala Leu Met Asn Ala Leu 245 250 255 Lys Asp Gin Pro Ala Arg Lys Ala Asn Val Glu lie Leu Glu lie Asp 260 265 270 Gly Val Ala Asn Val Met Arg Glu Arg Pro Glu Lys Val Ala Glu Ser 275 280 285 Val Gin Asn Phe Met Gin Gly Leu Gly Val Ala Ser Gly Ala Val Asn 290 295 300 Arg Arg Leu Ser Ala Thr Gly Asn Val Pro Lys lie Arg Asn Arg Ser 305 310 315 320 Ala Ser Met Asp Glu Tyr Asp Gin Pro Val Gly Val Lys Asn Leu lie 325 330 335 Tyr Asp Asn Thr Arg Arg Tyr Ser Lys Ala Thr Asp lie Pro Glu lie 340 345 350 Ser Glu Val Gly Asp His 355 <210> 11 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> up <400> 11 atgtcctccc agaaacctca gaaaattaca gtagatacta aatcatgcgg tcccctcact 60 gcatacctac acggaggtcg tgtaggaaaa gatgttgctg ctctcacagt tcatgatctc 120 ggatacaatg aattttttga atttgtaaat aacgagcaca tgagccagct cactcaacgc 180 gtattttgga ttcatgttga agtacctggg caaggggaca acatgcctga tctcccgtca 240 gactggaaat ttcctacaat gcaaaaaatc tcggagggta tctcagaact ttgtgatagc 300 cttgaattga aacatgtcgt tgttattggt gatggtgctg gcgcaaacat tgttgctcgg 360 cttgctatgg ctcgagaaga tatttgtctt ggagctattc ttattcactg cactggcaca 420 actgctggat tcatggagtc ccttcgggac cgtgtgaact catggaaact gaatacaatt 480 ggtatgcacc cgagtgtaga gaactatctg gtacttcatc gttttggtgt gtttataaaa 540 gctacaacag aagccgaact tcggggggct atcaagaatt tcctccaatc gctccgtgag 600 aacatcaatc ccaaaaatct caataagttt atccaagctt tcatggtgcg aacaaacatc 660 actgattcaa ttggaaactt gaaatgccca gtcctcttta tcacgggctc agttgcctcc 720 ttcaaccaca ccgtctacac gctctacaac gcccttatga atgctctgaa ggaccagccg 780 gcgcgtaagg ccaatgtgga gattcttgag attgacggag tcgccaatgt catgcgggaa 840 aggccggaga aagttgctga gtcggtgcag aacttcatgc agggattggg tgtagctagc 900 ggtgcggtca acagacgcct aagcgcaact ggaaacgttc caaagatccg caaccgctcg 960 gcatccatgg acgaatacga ccagccggtg ggagtaaaga atctcattta cgacaatacc 1020 cggcgctatt ccaaggccac tgacatacca gaaatctcgg aggtgggcga tcactaa 1077
Claims
1. A kit for detecting echinococcosis, characterized by, The kit comprises a protein as shown in SEQ ID NO:
10.
2. The kit of claim 1, wherein The kit is an indirect ELISA detection kit.
3. The kit of claim 2, 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.
4. A marker combination, characterized in that The marker combination comprises a protein as shown in SEQ ID NO: 10, and the marker combination further comprises one or more of Egr, antigen B and antigen 5.
5. Use of a protein as shown in SEQ ID NO: 10 or a marker combination of claim 4 in the preparation of a diagnostic agent for diagnosing a disease caused by Echinococcus granulosus.