CN115873090A - VAT1L protein fragment and application thereof in human echinococcosis - Google Patents

Abstract

The invention discloses a VAT1L protein fragment and application thereof in detection of human echinococcosis. The polypeptide comprises one or more of amino acid sequences shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. The invention also discloses a recombinant protein or a mutation thereof, wherein the recombinant protein comprises an amino acid sequence shown as SEQ ID NO. 6; the mutated amino acid sequence has at least 80% identity to 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%, echinococcosis can be effectively detected, the positive detection rate is 82%, the negative detection rate is 84%, and positive cases which cannot be detected by Egr can be detected.

Description

A VAT1L protein fragment and its application in human echinococcosis

technical field

The invention relates to the field of immunomedicine, in particular to a VAT1L protein fragment, a recombinant protein or its mutation, a nucleic acid sequence and its application in detecting or treating human echinococcosis.

Background technique

Hydatid disease, also known as echinococcosis, is a zoonotic disease, especially in central and western China (Tibet, Qinghai, Sichuan, Xinjiang, etc.) infection. Human echinococcosis is mainly divided into cystic echinococcosis and alveolar echinococcosis. Imaging detection is the most intuitive and commonly used diagnostic method for echinococcosis. However, due to the long incubation period of echinococcosis, and the cysts formed by echinococcosis can only be detected by imaging when the cysts formed by echinococcosis are large enough, serum Medical 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 these two antigens.

To date, several methods have been described for the laboratory diagnosis of echinococcosis, including the detection of antibodies, antigens, and cytokines. Among them, the development of antibody detection mainly depends on the development of hydatid antigen species. However, both natural purified antigens and recombinant purified antigens have certain deficiencies in specificity and/or sensitivity. More importantly, the etiology of human echinococcosis involves factors such as difficulty in sampling and separation of echinococcosis protein, and there have been no more antigens that can be used for serological detection.

The difficulties in developing antibodies against hydatid antigens in the prior art are as follows:

1) Extracting and identifying hydatid protein from hydatid cysts isolated after operation of hydatid patients is a technical difficulty in itself. If there are only a few identified echinococcin proteins, it will be more difficult to select proteins that may be used as echinococcin antigens.

2) There are few types of single antigens. Currently, the most widely used diagnostic antigens for echinococcosis are 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.

3) The commercial hydatid antigen is a natural purified antigen, which is isolated and purified from the broken soluble antigen fragment of Echinococcus granulosus, and is a mixture containing various hydatid proteins. The more proteins that are mixed, the more false-negative or false-positive results are likely to be generated in serum testing.

Contents of the invention

In order to solve the difficulty in developing antibodies against echinococcosis antigens in the prior art, the present invention provides a VAT1L protein fragment and its application in human echinococcosis. The present invention expands the echinococcosis antigen database by looking for more antigens that can be used to diagnose echinococcosis, so that these antigens may be single or combined to improve the specificity and sensitivity of laboratory diagnosis of echinococcosis, and also generate echinococcosis from echinococcosis A mature experimental procedure for extracting and identifying more hydatid proteins from the hydatid cysts isolated after surgery.

In order to solve the above technical problems, one of the solutions of the present invention provides a polypeptide, wherein the polypeptide comprises such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:4 and SEQ ID NO: One or more of the amino acid sequences shown in ID NO:5.

In order to solve the above technical problems, the second solution of the present invention provides a recombinant protein or its mutation, wherein, the recombinant protein comprises the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence of the mutation is the same as the The amino acid sequence of the recombinant protein has at least 80%, 85%, 90%, 95%, 98%, 99% identity and maintains the function of the recombinant protein.

In order to solve the above technical problems, the third solution of the present invention provides an isolated nucleic acid, wherein the isolated nucleic acid encodes the polypeptide described in one of the solutions of the present invention or the recombinant protein described in the second solution of the invention or its mutations. Preferably, the nucleotide sequence encoding the recombinant protein described in the second aspect of the invention is shown in SEQ ID NO:7.

In order to solve the above technical problems, the fourth aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid as described in the third aspect of the present invention.

In order to solve the above technical problems, the fifth aspect of the present invention provides a transformant, wherein it comprises the isolated nucleic acid as described in the third aspect of the present invention or the recombinant expression vector as described in the fourth aspect 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.

In order to solve the above-mentioned technical problems, the sixth solution of the present invention provides a kit for detecting echinococcosis, which includes the polypeptide described in one of the solutions of the present invention or the polypeptide described in the second solution of the present invention. The above-mentioned recombinant protein or its mutation.

Preferably, the kit is an indirect ELISA detection kit.

More preferably, the indirect ELISA detection kit also includes a secondary antibody, a coating solution, a washing solution, a color developing solution, a stop solution and a diluent.

In order to solve the above technical problems, the seventh solution of the present invention provides a marker combination, wherein the marker combination includes the polypeptide described in one of the solutions of the present invention or the recombinant protein or its mutation.

In a preferred embodiment, the marker combination further includes one or more of Egr, antigen B and antigen 5.

In order to solve the above technical problems, the eighth solution of the present invention provides an siRNA or mRNA vaccine against echinococcosis, wherein the siRNA or mRNA vaccine comprises and encodes the polypeptide or the present invention as described in one of the solutions of the present invention. The RNA sequence complementary to the recombinant protein or its mutated nucleotide sequence described in the second aspect of the invention.

In order to solve the above technical problems, 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 or its mutation described in the second aspect of the present invention, wherein the The method includes the following steps: cultivating the transformant according to 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.

In order to solve the above technical problems, the tenth scheme of the present invention provides a polypeptide as described in one of the schemes of the present invention or the recombinant protein or its mutation described in the second scheme of the present invention or the polypeptide described in the seventh scheme of the present invention The application of the combination of markers in the preparation of anti-hydatid antibodies or in the diagnosis of diseases caused by Echinococcus granulosus.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

(1) Found a new echinococcosis antigen VAT1L, which can be used to make ELISA kits and clinically detect human echinococcosis, and can complement each other with Egr antigen.

(2) The echinococcosis neoantigen can be purified in large quantities, which is convenient for large-scale screening of human echinococcosis.

Description of drawings

Figure 1 shows the results of SDS-PAGE and Western blotting of the whole protein extracted from the five cystic fluid fractions. The left picture shows the WB results of No.1 patient serum as the primary antibody and five cystic fluid components as antigens. The middle picture shows the WB results of normal Tibetan serum as the primary antibody and five cystic fluid components as antigens. The picture on the right shows the protein gel results of the five cystic fluid fractions. Experiments have shown that the cyst fluid whole protein has a strong immune response to the patient's serum, but basically no reaction to the normal Tibetan serum, and most of the protein bands that cause immune reactions are not high-abundance protein bands.

Figure 2 shows the purification results of the VAT1L recombinant protein. The figure shows the gradient elution results of the VAT1L recombinant protein, and the sample volume in each lane is 10 μl. Each lane in turn refers to the total bacterial protein after sonication, the flow-through after loading on the nickel column, and the elution gradient from 20mM imidazole to 1M imidazole.

Figure 3 shows the results of the WB verification experiment of the purified VAT1L recombinant protein. Numbers 1-9 refer to 9 patient sera taken before undergoing hydatid cystectomy. Control refers to the serum of normal Tibetans without disease. Egr_SDS-PAGE refers to the protein gel display of the commercialized antigen Egr, and the protein band indicated by the arrow is Antigen B.

Figure 4 shows the results of the ELISA experiment. VAT1L_OD(+) and Egr_OD(+) refer to the OD value of 607 cases of B-positive plasma, and VAT1L_OD(-) and Egr_OD(-) refer to the OD value of 636 cases of normal human plasma.

Fig. 5 is the ROC curve of VAT1L recombinant protein and commercialized antigen Egr. The picture on the left is the ROC curve of VAT1L. Criterion means that when VAT1L detects serum, it is positive if it is greater than 0.37 (echinococcosis), and less than or equal to 0.37 is negative (not affected by echinococcosis). The figure on the right is the ROC curve of Egr. Criterion means that when Egr detects serum, it is positive if it is greater than 0.99 (echinococcosis), and less than or equal to 0.99 is negative (not affected by echinococcosis).

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Example 1 The process of identifying more hydatid proteins from hydatid cysts isolated after operation of hydatid patients

1. First, the hydatid cysts separated from 10 hydatid patients were divided into four parts according to their tissue structure to extract protein. In the active stage, 10 hydatid cysts were divided into active hydatid cyst group and inactive hydatid cyst group.

2. Protein extraction was performed on the four components of each hydatid cyst, and the extracted protein was subjected to liquid enzymatic hydrolysis, followed by LC-MS/MS identification using QE mass spectrometer;

3. The original data of QE off-machine use maxquant protein identification software, use human and echinococcosis protein database collection, carry out echinococcosis protein identification, count the identification results and determine the hydatid cyst part with high echinococcin protein content, the results are shown in Table 1 and As shown in Table 2;

It can be seen from Table 1 and Table 2 that there are two basic conclusions: one is that the tissue parts with more hydatid protein identified are the two parts of the cyst fluid and protoscoleia; the other is that only the cyst fluid and The protein extracted from the protoscoleia part contained more hydatid protein.

Table 1 Protein identification results of active hydatid cyst group

_1 represents protocephala; _2 represents cystic fluid; _3 represents germinal layer; _4 represents corneal cortex.

Table 2 Protein identification results of inactive hydatid cyst group

_1 represents protocephala; _2 represents cystic fluid; _3 represents germinal layer; _4 represents corneal cortex

Example 2 Western Blot verification, gel cutting and enzymatic digestion and mass spectrometry identification (GeLC-MS/MS)

1. Next, according to the QE identification results of the cyst fluid and protoscoleum of 10 samples, the protoscoleum and cyst fluid components of five active hydatid cyst groups No.1-No.5 were selected for comparison with patient serum WB experiment in order to verify whether there is an immune reaction between the serum of hydatid patients and normal Tibetans and the extracted whole protein.

2. Due to the fact that the total amount of protein extracted from the protoscoleum fraction of some samples is very small, the Western Blotting experiment of the cystic fluid component protein was mainly carried out. The results are shown in Figure 1, taking No.1 patient serum as an example. , Immunoreactive profiles of sera from hydatid patients and normal Tibetans with five cystic fluid component proteins. The immunoreactivity profiles of other patients' sera and normal Tibetan sera to the 5 sample proteins were similar to those of No.1 patient.

3. According to the SDS-PAGE patterns of these five cystic fluid components, the SDS-PAGE gel was hydrolyzed by gel cutting and enzymatic hydrolysis against the immunological bands of WB, in order to be able to identify the higher molecular weight in the corresponding molecular weight segment through gel cutting separation. Polyhydatid protein, which increases the chance of finding hydatid antigens that immunoreact with sera. Additionally, this reduces the impact of high-abundance proteins on identification.

4. In order to reduce the loss and reduce the workload, we uniformly divided the 5 cystic fluid component proteins into 10 components according to the WB immune bands, and then each component was subjected to in-gel enzymatic hydrolysis.

5. After the LC-MS/MS analysis of the QE-HF-X mass spectrometer, the maxquant software used the same database as in Example 1 to carry out protein search and identification. The results are shown in Table 3.

6. Through data analysis, it can be seen that the hydatid proteins identified in the three samples of 1_2, 2_2 and 5_2 include the hydatid proteins identified in the other four samples. Therefore, the candidates for subsequent hydatid antigens are derived from this The echinococcin proteins identified jointly by each component of the three samples were looked for.

Table 3 Protein identification results of five cystic fluid fractions by enzymatic hydrolysis

Numbers in brackets indicate total identified proteins, while numbers outside brackets indicate hydatid proteins in each fraction. _2 represents cystic fluid.

Example 3 Screening of hydatid protein as candidate antigen

1. The common protein identified in the three samples of the same component was used as the candidate antigen library, and finally 93 unique proteins were generated from the 10 components.

2. The 93 proteins were first compared with the human protein database, and after deleting the proteins or protein fragments with a homology greater than 20% and those proteins that had been selected as hydatid antigens, the remaining proteins were identified as B cell antigens Epitope prediction.

Example 4 Epitope Prediction and Construction of Recombinant Plasmid

1. Antigen epitope prediction uses an online prediction tool http://tools.iedb.org/bcell/, imports the protein sequence, and selects the Bepipred Linear Epitope Prediction 2.0 prediction method to get information about the possible antigenic epitope of this protein sequence list.

2. According to the epitope list of each candidate protein, select sequence fragments containing one or more epitopes to form the sequence of the recombinant protein, and import this sequence into the comparison tool for comparison with the human protein database. Sequence fragments with human protein similarity ≤ 20% are selected to construct recombinant proteins.

3. The final 14 protein sequence fragments were inserted into the pET-30a(+) plasmid by molecular cloning technology to construct a recombinant plasmid, and the fragments were confirmed to be inserted by sequencing.

Expression and purification of embodiment five recombinant proteins

1. Transfer the recombinant plasmid into BL21(DE3) for small-scale test expression, confirm that the protein can be expressed and the molecular weight is correct, and then expand the culture.

2. After the collected bacterial liquid is ultrasonically extracted, the protein is then purified by a nickel column through the His-tag carried by the plasmid. Figure 2 shows the purification result of the recombinant protein constructed from the VAT1L fragment of the present invention.

Example 6 Western Blotting Verification of VAT1L Recombinant Protein

1. Eight of the 14 recombinant proteins were successfully expressed. Therefore, the purified 8 recombinant proteins were subjected to Western Blotting experiments with 9 postoperative patient sera, and the results showed that only 4 recombinant proteins had a large immune reaction with the patient sera. Contains the VAT1L recombinant protein, and then uses the VAT1L recombinant protein as a hydatid antigen candidate to conduct ELISA verification tests on more patient sera.

2. Figure 3 shows the WB of VAT1L recombinant protein and commercialized antigen Egr (product name: hydatid purified antigen Echinococcus granulosus, article number: YM-VI08, supplier: Hangzhou Yiminuo Biotechnology Co., Ltd.) and 9 patient sera The results of the experiment showed that the immune reaction rate of the VAT1L recombinant protein was 56%, and the immune reaction rate of the commercialized antigen Egr was 67% (only the samples with obvious bands between 43KD and 34KD were counted), and the VAT1L recombinant protein detected No. 1 positive patient, while Egr was not detected. Table 4 shows the B cell antigen epitope, nucleotide sequence and amino acid sequence of the VAT1L recombinant protein.

Table 4 B cell antigen epitope, nucleotide sequence and amino acid sequence of VAT1L recombinant protein

ELISA experimental verification of a kind of echinococcosis recombinant antigen VAT1L recombinant protein of embodiment seven

The detected objects were 607 cases of B-ultrasound-positive plasma (including suspected cases) and 636 cases of normal human plasma (determined as negative), which were carried out according to the standard operating procedures of indirect ELISA. The results showed that the positive detection rate of VAT1L recombinant protein was 82%. The negative detection rate was 84%, while the positive detection rate was 86% and the negative detection rate was 92% when the commercial antigen Egr was used to detect the same plasma.

1. ELISA experiment specific operation steps

(1) Antigen quantification: Each antigen was blown evenly with a pipette gun before coating, and a micro-ultraviolet spectrophotometer was used to quantify the antigen to ensure that the protein was not degraded. There is no absorption peak at A280, which means that the amount of antigen is very low and it is not suitable for coating; if the protein is precipitated or insoluble, add 8M urea dropwise, blow evenly, and centrifuge to take the supernatant to measure the concentration.

(2) Coating: compare the concentration of the antigen tube wall and the measured value, and take the lower value; the coating amount is 2 μg/ml, 10ml/plate, and the amount of coated antigen is prepared according to the actual usage; the coated ELISA plate is marked: 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 marking the ELISA plate, add the coating antigen, 100 μl/well, and coat at 4°C overnight or at 37°C for 2h.

(3) Plate washing: wash the plate once with a plate washer, and pat dry on absorbent paper. The formula of 50× lotion is: 154.4g of Tris; 149.0g of NaCl; 24.0ml of Tween-20; 800ml of pure water; about 45ml of concentrated hydrochloric acid to adjust the pH to 7.2, and the volume of pure water to 1000ml. Store at 4°C.

(4) Blocking: 2% skimmed milk powder was used as a blocking solution for blocking, 200 μl/well, at 4° C. overnight or at 37° C. for 2 hours.

(5) Plate washing: After sealing the plate, wash the plate once with a plate washer, and pat dry on absorbent paper.

(6) Add primary antibody: add plasma samples, dilute the plasma with PBS at a volume ratio of 1:500, and incubate at 37°C for 1 hour.

(7) Plate washing: Wash the plate for more than 3 times with a plate washer, and pat dry on absorbent paper.

(8) Add secondary antibody (manufacturer: Beyont; product number: A0201): Since the source of the primary antibody is human plasma, the secondary antibody is added with goat anti-human 1:500, and incubated at 37°C for 1 hour.

(9) Plate washing: wash the plate more than 3 times with a plate washer, and pat dry on absorbent paper. While washing the plate, prepare the chromogenic solution (TMB).

(10) Color development: After preparing the stop solution, add 100 μl/well of color development solution, shake the plate to speed up the color development process and pay close attention to observation.

(11) TMB color development: when the color development reaches 4 minutes (generally, the blank and negative color development should not be too high), add 50 μl/well stop solution, and wait for 10 minutes after adding the stop solution, so that the stop is complete and the color is uniform (you can shake it) board accelerates the termination process), reading after termination

(12) Reading: The microplate reader must be preheated for more than 30 minutes; TMB color detection wavelength: 450nm. Open the corresponding microplate reader measurement software and read. Store the data to the specified location and complete the data.

(13) Matters needing attention:

When adding samples to the 96-well plate with a gun, pay attention to avoid air bubbles and prevent the added samples from hanging on the wall.

After adding the sample, observe the whole plate to ensure that there are no air bubbles at the bottom of the well; when air bubbles are found, shake the plate or use a pipette tip to remove the air bubbles.

Close attention should be paid when washing the plate to ensure that the washing plate is complete and thorough.

Close attention should be paid to the color development, and the color development of the negative, blank, and positive controls should be combined to stop in time.

(14) Various reagent formulas

Coating solution: sodium carbonate-sodium bicarbonate buffer solution, pH9.6: 1.59g Na ₂ CO ₃ , 2.93g NaHCO ₃ , dilute to 1000ml with pure water; finally check the pH value with pH test paper. Store at 4°C.

2. Analysis of ELISA results

(1) Negative and positive judgment criteria: select the mean (X) and standard deviation (SD) of negative serum samples, and the upper limit cut-off value of the confidence interval is X+2SD. If the OD value of the sample to be tested at 450nm is greater than or equal to X+2SD, it can be judged as positive, and if it is less than X+2SD, it can be judged as negative.

(2) ROC curve: MedCalc software (version 19.4.0) was used to draw the ROC curve (Receiver Operating Characteristic curve) to evaluate the optimal detection efficiency of the antigen.

(3) by calculation, using the echinococcosis antigen of the present invention to immunize 607 cases of B-ultrasonic positive plasma (containing suspected) and 636 cases of normal human plasma (determined as negative), its sensitivity is 82%, and specificity is 84%; The sensitivity was 86% and the specificity was 92% when the commercial antigen Egr was used to detect the same plasma. Figure 4 shows the box plot of the detected OD values of the VAT1L recombinant protein and the commercialized antigen Egr.

(4) Figure 5 shows the ROC curves of VAT1L recombinant protein and commercialized antigen Egr. Although the specificity of VAT1L recombinant protein is lower than that of commercial antigen Egr, the sensitivity of VAT1L recombinant protein is basically the same as that of commercial antigen Egr, and the dual characteristics of VAT1L recombinant antigen and commercial antigen Egr are simulated by logistic regression of MedCalc software The ROC curve shows that the detection efficiency of the two antigens together is very good. It shows that the VAT1L recombinant antigen may be used as a supplement to the commercialized antigen, and the samples that have not been tested positive by the commercialized antigen can be retested, so the VAT1L recombinant protein can be used as a candidate for the neoantigen of echinococcosis.

SEQUENCE LISTING

<110> Shenzhen Huada Life Science Research Institute

<120> A VAT1L protein fragment and its application in human echinococcosis

<130> P22012954C

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<170> PatentIn version 3.5

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Ser Val Asp Glu Lys Thr Ala Ile

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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 (10)

1. A polypeptide comprising one or more of the amino acid sequences shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5.

2. 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%, 99% identity to the amino acid sequence of the recombinant protein and maintains the function of the recombinant protein.

3. An isolated nucleic acid encoding the polypeptide of claim 1 or the recombinant protein of claim 2 or a mutation thereof; preferably, the nucleotide sequence encoding the recombinant protein of claim 2 is as set forth in SEQ ID NO 7.

4. A recombinant expression vector comprising the isolated nucleic acid of claim 3.

5. A transformant comprising the isolated nucleic acid of claim 3 or the recombinant expression vector of claim 4, wherein the transformant is a bacterium or a eukaryotic cell; preferably, the bacterium is e.coli BL21; more preferably, the transformant expresses an amino acid sequence shown as SEQ ID NOS: 1 to 6.

6. A kit for detecting echinococcosis, comprising the polypeptide of claim 1 or the recombinant protein of claim 2 or a mutation thereof;

preferably, the kit is an indirect ELISA detection kit;

more preferably, the indirect ELISA detection kit further comprises a second antibody, a coating solution, a washing solution, a developing solution, a stop solution, and a diluent.

7. A marker combination comprising the polypeptide of claim 1 or the recombinant protein of claim 2 or a mutation thereof;

preferably, the marker combination further comprises one or more of Egr, antigen B and antigen 5.

8. An siRNA or mRNA vaccine against echinococcosis comprising an RNA sequence complementary to a nucleotide sequence encoding the polypeptide of claim 1 or the recombinant protein of claim 2 or a mutation thereof.

9. A method for producing the polypeptide of claim 1 or the recombinant protein of claim 2 or a mutation thereof, wherein the transformant of claim 5 is cultured under conditions suitable for fermentation thereof to express the polypeptide or the recombinant protein or the mutation thereof.

10. Use of the polypeptide of claim 1 or the recombinant protein of claim 2 or the mutation thereof or the marker combination of claim 7 for the preparation of anti-echinococcosis antibodies or diagnostic agents for the diagnosis of diseases caused by echinococcosis granulosa.

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