A vat1l protein fragment and application thereof in human hydatidosis

Abstract

The application discloses a VAT1L protein fragment and application thereof in human echinococcosis detection. 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 and SEQ ID NO:5. The application further discloses a recombinant protein or a mutation thereof, wherein the recombinant protein comprises an amino acid sequence shown in SEQ ID NO:6; the amino acid sequence of the mutation has at least 80% identity with the amino acid sequence of the recombinant protein and maintains the function of the recombinant protein. The immunoreaction rate of the VAT1L recombinant protein is 56%, the echinococcosis can be effectively detected, the positive detection rate is 82%, the negative detection rate is 84%, and the positive cases that cannot be detected by Egr can be detected.

Description

Technical Field

[0001] This invention relates to the field of immunomedicine, and in particular to a VAT1L protein fragment, recombinant protein or its mutation, nucleic acid sequence and its application in 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] Several methods for the laboratory diagnosis of echinococcosis have been described to date, including the detection of antibodies, antigens, and cytokines. The development of antibody detection has primarily relied on the development of echinococcosis antigens. However, both naturally purified and recombinant purified antigens have limitations in specificity and / or sensitivity. More importantly, human echinococcosis involves difficulties in sampling and isolating echinococcosis proteins, resulting in a lack of more antigens available for serological detection.

[0004] The difficulties in developing antibodies against echinococcosis antigens in the existing technology are as follows:

[0005] 1) 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 will be even more difficult.

[0006] 2) Limited number of single antigens. Currently, the most widely used diagnostic antigens for echinococcosis are antigen B and antigen 5. Many studies have shown that these two antigens can produce a certain number of false positives or false negatives in serum testing.

[0007] 3) 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

[0008] To address the difficulties in developing antibodies against echinococcosis antigens in existing technologies, this invention provides a VAT1L protein fragment and its application in 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. It also provides a mature experimental procedure for extracting and identifying more echinococcosis proteins from echinococcosis cysts isolated from patients after surgery.

[0009] To solve the above-mentioned technical problems, one of the solutions of the present invention provides a polypeptide, wherein the polypeptide comprises one or more amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0010] To solve the above-mentioned technical problems, a second solution 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: 6; the mutated amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, and 99% identity with the amino acid sequence of the recombinant protein, and maintains the function of the recombinant protein.

[0011] To address the aforementioned technical problems, a third aspect of the present invention provides an isolated nucleic acid, wherein the isolated nucleic acid encodes a polypeptide as described in a first aspect of the present invention or a recombinant protein or a mutation thereof as described in a second aspect of the present invention. Preferably, the nucleotide sequence encoding the recombinant protein as described in a second aspect of the present invention is shown in SEQ ID NO: 7.

[0012] To solve the above-mentioned technical problems, the fourth solution of the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid as described in the third solution of the present invention.

[0013] To address the aforementioned technical problems, a fifth embodiment of the present invention provides a transformant comprising the isolated nucleic acid as described in a third embodiment of the present invention or the recombinant expression vector as described in a fourth embodiment of the present invention, wherein the transformant is a bacterium or a eukaryotic cell. Preferably, the bacterium is E. coli BL21. More preferably, the transformant expresses the amino acid sequence shown in SEQ ID NO: 1-6.

[0014] To address the aforementioned technical problems, a sixth aspect of the present invention provides a kit for detecting echinococcosis, the kit comprising a polypeptide as described in a first aspect of the present invention or a recombinant protein or a mutation thereof as described in a second aspect of the present invention.

[0015] Preferably, the kit is an indirect ELISA detection kit.

[0016] More preferably, the indirect ELISA detection kit further includes a second antibody, coating solution, washing solution, chromogenic solution, stop solution, and diluent.

[0017] To solve the above-mentioned technical problems, the seventh embodiment of the present invention provides a combination of biomarkers, wherein the combination of biomarkers includes a polypeptide as described in the first embodiment of the present invention or a recombinant protein or a mutation thereof as described in the second embodiment of the present invention.

[0018] In a preferred embodiment, the biomarker combination further includes one or more of Egr, antigen B, and antigen 5.

[0019] To solve the above-mentioned technical problems, the eighth embodiment 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 a polypeptide as described in the first embodiment of the present invention or a recombinant protein as described in the second embodiment of the present invention or a mutated nucleotide sequence thereof.

[0020] To solve the above-mentioned technical problems, the ninth aspect of the present invention provides a method for producing a polypeptide as described in the first aspect of the present invention or a recombinant protein or its mutation as described in the second aspect of the present invention, wherein the method includes the following steps: culturing a transformant as 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 its mutation.

[0021] To address the aforementioned technical problems, the tenth aspect of this invention provides the application of a polypeptide as described in the first aspect of this invention, a recombinant protein as described in the second aspect of this invention, or a combination of its mutations or biomarkers as described in the seventh aspect of this invention, in the preparation of anti-echinococcosis antibodies or in diagnostic agents for diagnosing diseases caused by Echinococcus granulosus.

[0022] 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.

[0023] The reagents and raw materials used in this invention are all commercially available.

[0024] The positive and progressive effects of this invention are as follows:

[0025] (1) A new echinococcosis antigen, VAT1L, was found that can be used to make ELISA kits and for clinical detection of human echinococcosis, and can complement Egr antigen.

[0026] (2) This new echinococcosis antigen can be purified in large quantities, which facilitates large-scale screening of human echinococcosis. Attached Figure Description

[0027] Figure 1 The SDS-PAGE and Western blotting results of the total 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 normal Tibetan serum as the primary antibody and the five cyst fluid fractions as antigens. The right image shows the protein gel extraction results of the five cyst fluid fractions. The experiment demonstrates that the total proteins from the cyst fluid elicit a strong immune response to patient serum, but show virtually no response to normal Tibetan serum, and most of the protein bands that elicit an immune response are not high-abundance protein bands.

[0028] Figure 2 The purification results of the VAT1L recombinant protein are shown in the figure. The gradient elution results of the VAT1L recombinant protein are shown in the figure, with a sample volume of 10 μl in each lane. Each lane represents, in turn, the total 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.

[0029] Figure 3 This is a Western blot (WB) validation experiment of the purified VAT1L 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 results of the commercial antigen Egr; the protein band indicated by the arrow represents Antigen B.

[0030] Figure 4 This is a presentation of ELISA test results. VAT1L_OD(+) and Egr_OD(+) refer to the OD values ​​of 607 ultrasound-positive plasma samples, while VAT1L_OD(-) and Egr_OD(-) refer to the OD values ​​of 636 normal plasma samples.

[0031] Figure 5 The figures show the ROC curves for recombinant VAT1L protein and the commercial antigen Egr. The left figure shows the ROC curve for VAT1L; the Criterion indicates that a VAT1L Criterion greater than 0.37 in serum testing is positive (indicating echinococcosis), and less than or equal to 0.37 is negative (indicating no echinococcosis). The right figure shows the ROC curve for Egr; the Criterion indicates that an Egr Criterion greater than 0.99 in serum testing is positive (indicating echinococcosis), and less than or equal to 0.99 is negative (indicating no echinococcosis). Detailed Implementation

[0032] 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.

[0033] Example 1: The process of identifying more hydatid proteins from hydatid cysts isolated from patients after surgery.

[0034] 1. First, the hydatid cysts isolated from 10 patients with hydatid cysts after surgery were divided into four parts according to their tissue structure for protein extraction. From the inside out, these parts were the protocercariae, cyst fluid, germinal layer, and keratinocytes. Then, based on whether they were in the active phase, the 10 hydatid cysts were divided into an active hydatid cyst group and an inactive hydatid cyst group.

[0035] 2. Proteins were extracted from all four components of each echinococcosis cyst, and the extracted proteins were subjected to liquid enzymatic digestion, followed by LC-MS / MS identification using QE mass spectrometry;

[0036] 3. The raw data from the QE machine were analyzed using the maxquant protein identification software and a combination of human and echinococcosis protein databases. The results were statistically analyzed and the echinococcosis cysts with high echinococcosis protein content were identified. The results are shown in Tables 1 and 2.

[0037] Tables 1 and 2 reveal two key conclusions: First, the tissues from which more hydatid proteins were identified were the cyst fluid and the protocercariae; second, only the cyst fluid and protocercariae extracted from the active hydatid cysts contained more hydatid proteins.

[0038] Table 1. Protein identification results of the active echinococcosis cysts

[0039]

[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 protocephalan; _2 represents the cyst fluid; _3 represents the germinal layer; _4 represents the keratinocyte.

[0044] Example 2: Western blotting verification, 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, the protocercariae and cyst fluid components of five active echinococcosis cyst groups No.1-No.5 were selected for Western blot (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 total protein.

[0046] 2. Because the total protein content extracted from the protocercariae of some samples was very low, Western blotting experiments were mainly performed on the proteins in the cyst fluid. 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 spectra of these five cyst fluid components, the SDS-PAGE gel was digested using enzymatic digestion, comparing it with the immunohistochemical bands obtained from Western blotting. This was intended to separate and identify more echinococcosis proteins in the corresponding molecular weight ranges, increasing the probability of finding echinococcosis antigens that react with serum. Additionally, this method can reduce the impact of high-abundance proteins on identification.

[0048] 4. To reduce losses and workload, we uniformly divided the five cyst fluid components into 10 components based on the WB immune bands, and then each component was subjected to in-gel enzymatic hydrolysis.

[0049] 5. After LC-MS / MS analysis using the QE-HF-X mass spectrometer, protein library identification was performed using the same database as in Example 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 candidate echinococcosis antigens should be searched from the echinococcosis proteins that were jointly 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 of echinococcosis proteins as candidate antigens

[0055] 1. Common proteins identified in the same component in three samples were used as a candidate antigen library, 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. Proteins or protein fragments with homology greater than 20% and those proteins that had been selected as echinococcosis antigens were removed. The remaining proteins were then used to predict B-cell antigenic epitopes.

[0057] Example 4: Antigenic Epitope Prediction and Recombinant Plasmid Construction

[0058] 1. Antigenic epitope prediction used an online prediction tool: http: / / tools.iedb.org / bcell / . By importing the protein sequence and selecting the Bepipred Linear Epitope Prediction 2.0 prediction method, a list of sequences that the protein may become an antigenic epitope can be obtained.

[0059] 2. Based on the list of antigenic epitopes for each candidate protein, select sequence fragments containing one or more epitopes to form the recombinant protein sequence, and import this sequence into an alignment tool for comparison with human protein databases. Only sequence fragments with ≤20% similarity to human proteins are selected for constructing recombinant proteins.

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

[0061] Example 5: Expression and Purification of Recombinant Proteins

[0062] 1. Transform the recombinant plasmid into BL21(DE3) for small-scale expression. After confirming that the protein can be expressed and that the molecular weight is correct, then proceed with amplification.

[0063] 2. After the collected bacterial culture is subjected to ultrasonic protein extraction, it is then purified using a nickel column using a His-tag carried by a plasmid. Figure 2 The purification results of the recombinant protein constructed from the VAT1L fragment of this invention are shown.

[0064] Example 6: Western Blotting Validation of VAT1L Recombinant Protein

[0065] 1. Eight out 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 showed a strong immune response to the patient serum, including the VAT1L recombinant protein. The VAT1L recombinant protein was subsequently used as a candidate echinococcosis antigen for ELISA validation experiments with serum from more patients.

[0066] 2. Figure 3Western blot (WB) results of recombinant VAT1L protein and commercial antigen Egr (product name: purified Echinococcus granulosus antigen, catalog number: YM-VI08, supplier: Hangzhou Yiminuo Biotechnology Co., Ltd.) with serum from 9 patients are presented. The results show that the immunoreactivity rate of recombinant VAT1L protein is 56%, and the immunoreactivity rate of commercial antigen Egr is 67% (only samples with a clear band between 43 KD and 34 KD are counted). Recombinant VAT1L protein was detected in patient 1 who was positive, while Egr was not detected. Table 4 shows the B-cell antigenic epitopes, nucleotide sequence, and amino acid sequence of recombinant VAT1L protein.

[0067] Table 4. B-cell antigenic epitopes, nucleotide sequences, and amino acid sequences of VAT1L recombinant protein.

[0068]

[0069] Example 7: ELISA assay verification of a recombinant echinococcosis antigen VAT1L 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 VAT1L recombinant protein was 82%, and the negative detection rate was 84%. When the same plasma was tested with the commercial antigen Egr, 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, 8 M 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 10ml / 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 2 h at 37℃.

[0074] (3) Plate washing: After coating, wash the plates once with a plate washer and pat dry on absorbent paper. The 50× washing solution formula is: 154.4 g Tris; 149.0 g NaCl; 24.0 ml Tween-20; 800 ml pure water; adjust the pH to 7.2 with approximately 45 ml 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 mixture is blocked overnight at 4℃ or blocked for 2 hours at 37℃.

[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 plasma sample, dilute plasma with PBS at a volume ratio of 1:500, and incubate at 37°C for 1 h.

[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; catalog 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 h.

[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 4 min (generally, the color development of blank and negative should not be too high), add 50 μl / well of stop solution. After adding the stop solution, let it stand for 10 min 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 software and read the value. 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 450 nm 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 ultrasound-positive plasma (including suspected cases) and 636 normal plasma (confirmed negative) with the echinococcosis antigen of the present invention was 82% and the specificity was 84%; while the sensitivity of detecting the same plasma with the commercial antigen Egr was 86% and the specificity was 92%. Figure 4 Box plots showing the detection OD values ​​of VAT1L recombinant protein and commercial antigen Egr are presented.

[0095] (4) Figure 5 The ROC curves of recombinant VAT1L protein and the commercial antigen Egr are shown. Although the specificity of recombinant VAT1L protein is lower than that of commercial antigen Egr, its sensitivity is roughly equivalent to that of commercial antigen Egr. Furthermore, logistic regression simulation using MedCalc software to obtain the dual-characteristic ROC curves of recombinant VAT1L antigen and commercial antigen Egr shows that the detection efficacy of the two antigens together is excellent. This indicates that recombinant VAT1L antigen can likely serve as a supplement to commercial antigen for retesting samples that were not detected positive by the commercial antigen. Therefore, recombinant VAT1L protein can be considered a candidate for a new echinococcosis antigen. SEQUENCE LISTING <110> Shenzhen BGI Life Science Research Institute <120> A VAT1L protein fragment and its application in human echinococcosis <130> P22012954C <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Table 1 <400> 1 Ser Val Asp Glu Lys Thr Ala Ile 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Tablet 2 <400> 2 His Ala Cys Thr Tyr Ile His Thr Arg Ser Arg Ala Arg Val Arg Gly 1 5 10 15 Cys <210> 3 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Tablet 3 <400> 3 Leu Val Ser Phe Leu Ser Leu Ser Arg Leu Ile Asp Phe Ser Phe Ile 1 5 10 15 Ala Met Thr Thr Glu Gly Glu Gly Thr Asn Pro Pro Val Ala Gly Ala 20 25 30 Asn Ala Pro Glu Gly Glu Gly Ser Lys Glu Thr Glu Ala Pro Thr Thr 35 40 45 Ser Ala Pro Pro Val Lys Gln Thr Lys Cys Val Leu Leu Thr 50 55 60 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Epitope 4 <400> 4 Asp Gln Arg Thr Val Gly Lys Gly Glu Ile Ala Val Glu Val Glu Ala 1 5 10 15 Cys Gly Val Gly Phe Gln Asp Leu Met Met Arg Gln Gly Leu 20 25 30 <210> 5 <211> 40​​​​​​​​​​​​​​​​​​​​​​​​​​​<212> PRT <213> Artificial Sequence <220> <223> Recombinant protein of VAT1L <400> 6 Met Ala His Gln Ser Val Asp Glu Lys Thr Ala Ile Glu Pro Val Ile 1 5 10 15 Thr Ala His Ala Cys Thr Tyr Ile His Thr Arg Ser Arg Ala Arg Val 20 25 30 Arg Gly Cys Leu Ser Pro Pro Leu Pro Val Leu Val Ser Phe Leu Ser 35 40 45 Leu Ser Arg Leu Ile Asp Phe Ser Phe Ile Ala Met Thr Thr Glu Gly 50 55 60 Glu Gly Thr Asn Pro Pro Val Ala Gly Ala Asn Ala Pro Glu Gly Glu 65 70 75 80 Gly Ser Lys Glu Thr Glu Ala Pro Thr Thr Ser Ala Pro Pro Val Lys 85 90 95 Gln Thr Lys Cys Val Leu Leu Thr Gly Phe Gly Gly Pro Lys Tyr Leu 100 105 110 Arg Val Gln Leu Lys Asp Gln Arg Thr Val Gly Lys Gly Glu Ile Ala 115 120 125 Val Glu Val Glu Ala Cys Gly Val Gly Phe Gln Asp Leu Met Met Arg 130 135 140 Gln Gly Leu Leu Asp Phe Leu Gly Lys Pro Pro Phe Val Met Gly Ser 145 150 155 160 Glu Cys Cys Gly Lys Val Ala Glu Val Gly Glu Gly Val Thr Lys Phe 165 170 175 Lys Val Gly Asp Glu Val Ile Val Leu Cys Asp Asn Gly Ala Trp Thr 180 185 190 Glu His Leu Val Val Arg Ala Ile Pro Glu Glu Pro Ser Glu Ser Gly 195 200 205 Ala Glu His Met Glu Ala Pro Gly Asn Ala Ala Pro Leu Ala Leu Val 210 215 220 Leu His Lys Pro Thr Ser Leu Ser Ala Asn Gln Ala Ala Ala Phe Leu 225 230 235 240 <210> 7 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Editing the nucleic acid sequence of VAT1L <400> 7 atggcccacc aatcagtgga cgagaagaca gcgatagagc ctgtgattac cgctcatgca 60 tgcacctaca tacacacacg ttcacgcgct cgtgtccgag gttgtctctc accacctctg 120 cccgttcttg ttagttttct ctctctttca cgtctgatcg acttttcttt catcgcaatg 180 accactgaag gtgaaggtac caacccccct gttgctggag ccaacgctcc agagggtgaa 240 ggcagcaagg aaactgaagc accaaccaca tcagcacctc cagtgaagca aacgaaatgc 300 gtccttctca ccgggttcgg tgggcccaag taccttcgtg ttcagttgaa ggaccaaagg 360 actgtcggaa aaggcgaaat tgctgtagag gtggaagcct gtggtgttgg atttcaggac 420 ttgatgatgc gtcagggact gcttgatttt ctcggcaagc cccccttcgt tatgggcagc 480 gagtgctgtg gaaaggttgc tgaagttggt gaaggggtga cgaaatttaa ggttggtgac 540 gaagtaatcg tgctttgcga caatggagcc tggacggagc acctagtggt tcgagctatt 600 ccagaagagc caagtgaaag tggagctgag cacatggagg ctcccggcaa cgcagcaccg 660 ttagctttgg tcctgcacaa acccacctca ctatcagcca atcaggctgc cgcattcctc 720 taa 723

Claims

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

6.

2. An isolated nucleic acid, comprising, The isolated nucleic acid encodes the recombinant protein as claimed in claim 1.

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

7.

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

5. A transformant characterized in that, It comprises the isolated nucleic acid as claimed in claim 2 or 3 or the recombinant expression vector as claimed in 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 amino acid sequence as shown in SEQ ID NO:

6.

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

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 transformant as claimed in any one of claims 5-7 is cultured under conditions suitable for its fermentation to express the recombinant protein.

14. Use of the recombinant protein as claimed in claim 1 or the marker combination as claimed in 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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