CN115850424A - UP protein fragment and application thereof in human echinococcosis - Google Patents

Abstract

The invention discloses a UP protein fragment and application thereof in 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, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9. The invention also discloses the application of the polypeptide or the marker combination in preparing anti-echinococcosis antibodies or diagnostic agents for diagnosing diseases caused by echinococcosis granulosa. The echinococcus neoantigen UP can be used for manufacturing an ELISA kit and clinically detecting the human echinococcosis; and the human source echinococcosis can be purified in large quantity, and is convenient to screen in large scale.

Description

UP protein fragment and application thereof in human echinococcosis

Technical Field

The invention belongs to the field of immune medicine, and particularly relates to a polypeptide, namely a human echinococcosis neo-antigen UP protein fragment, a nucleic acid sequence and application thereof in preparation of a medicament for detecting or treating human echinococcosis.

Background

Echinococcosis, also known as echinococcosis, is a disease of both humans and animals, and is especially serious in the middle and western regions (Tibet, qinghai, sichuan, xinjiang, etc.) of China. Human echinococcosis is mainly classified into cystic echinococcosis and alveolar echinococcosis. The imaging detection is the most intuitive and commonly used echinococcosis diagnosis method, but because the incubation period of echinococcosis is long and the echinococcosis can be detected by imaging only when the cysts formed by echinococcosis are large to a certain extent, the serological detection is one of the important auxiliary means for imaging diagnosis of echinococcosis. Currently, the serological diagnostic antigens that can be used to diagnose echinococcosis are mainly Antigen B (Antigen B) and Antigen5 (Antigen 5) and some recombinant antigens related to these two antigens.

Extraction and identification of hydatid protein from hydatid cysts separated from hydatid patients after operation are a technical difficulty. If the identified hydatid proteins are of very few species, it is more difficult to select from those proteins that are likely to be antigens of hydatid.

Secondly, there are few single antigen species. The currently most widely used diagnostic antigens for echinococcosis are Antigen B and Antigen5, and many studies show that the two antigens generate certain false positive or false negative in serum detection.

The commercial echinococcus antigen is a natural purified antigen, and a soluble antigen fragment isolated and purified from the crushed echinococcus granulosus is a mixture comprising a plurality of echinococcus proteins. The more proteins mixed the more likely it is to produce more false negative or false positive results in serum testing.

Disclosure of Invention

In order to solve the technical defects of difficult extraction and identification of echinococcosis protein, few echinococcosis antigens and the like in the prior art, the invention provides a polypeptide, namely a human echinococcosis new antigen UP protein fragment, a nucleic acid sequence and application thereof in preparing a medicament for detecting or treating human echinococcosis. The invention expands the infusorian antigen database by searching more antigens which can be used for diagnosing the echinococcosis, so that the antigens, singly or in combination, can possibly improve the specificity and the sensitivity for diagnosing the echinococcosis in a laboratory.

In a first aspect, the invention provides a polypeptide, wherein the polypeptide comprises 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, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.

In a second aspect, the invention provides an isolated nucleic acid, wherein the isolated nucleic acid encodes a polypeptide according to the first aspect of the invention.

In a third aspect, the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid of the second aspect of the invention.

In a fourth aspect, the invention provides a transformant comprising the isolated nucleic acid of the second aspect or the recombinant expression vector of the third aspect, wherein the transformant is a bacterium or a eukaryotic cell.

In some preferred embodiments, the bacterium is e.

In a fifth aspect, the invention provides a kit for detecting echinococcosis, wherein the kit comprises a polypeptide according to the first aspect of the invention.

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

In some more preferred embodiments, the indirect ELISA detection kit further comprises a second antibody, a coating solution, a washing solution, a developing solution, a stop solution, and a diluting solution.

In a sixth aspect, the present invention provides a marker combination comprising a polypeptide according to the first aspect of the invention.

In some preferred embodiments, the marker combination further comprises one or more of Egr, antigen B and antigen 5.

In a seventh aspect, the invention provides an siRNA or mRNA vaccine against echinococcosis, wherein the siRNA or mRNA vaccine comprises an RNA sequence complementary to a nucleotide sequence encoding a polypeptide according to the first aspect of the invention.

In an eighth aspect, the invention provides a method of producing a polypeptide according to the first aspect of the invention, wherein the method comprises the steps of: the transformant according to the fourth aspect of the present invention is cultured under conditions suitable for fermentation thereof to express the polypeptide.

According to a ninth aspect of the present invention, there is provided a use of the polypeptide of the first aspect of the present invention or the marker combination of the sixth aspect of the present invention for preparing an anti-echinococcosis antibody or a diagnostic agent for diagnosing a disease caused by echinococcosis granulosa.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

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

The positive progress effects of the invention are as follows:

(1) Provides a neoantigen UP of hydatid which can be used for manufacturing an ELISA kit and used for clinically detecting human echinococcosis; and can be used as a mutual supplement with the Egr antigen to carry out more sensitive detection on the echinococcosis.

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

Drawings

FIG. 1 shows the SDS-PAGE and Western blotting results of whole proteins extracted from five cyst fluid fractions. The left panel is shown with WB results with the serum of patient No.1 as primary antibody and five cyst fluid fractions as antigens. The panel is shown with WB results with normal Tibetan human serum as primary antibody and five cyst fluid components as antigens. The right panel is a display of the protein gel results for the five cyst fluid components.

FIG. 2 is a display of the purification results of UP recombinant protein. The figure shows the result of gradient elution of the UP recombinant protein, and the amount of the sample loaded in each lane was 10. Mu.l. Each lane in turn refers to the bacterial holoprotein after sonication, the flow-through after loading on a nickel column, and the elution gradient from 20mM imidazole to 1M imidazole.

FIG. 3 shows WB validation experiment results of the purified UP recombinant protein. The numbers 1-9 refer to the serum taken from 9 patients before hydatid cyst removal. Control refers to normal Tibetan human serum that is not diseased. Egr _ SDS-PAGE refers to the protein gel result display of commercial Antigen Egr, and the protein band indicated by the arrow is Antigen B.

FIG. 4 shows the result of ELISA experiment. UP _ OD (+) and Egr _ OD (+) refer to the OD values of 607 cases of B-type ultrasound-positive plasma, UP _ OD (-) and Egr _ OD (-) refer to the OD values of 636 cases of normal human plasma.

FIG. 5 is a ROC curve of UP recombinant protein and commercial antigen Egr. The left graph is ROC curve of UP, criterion means that UP is positive (suffering from echinococcosis) when detected by serum, and is negative (not suffering from echinococcosis) when greater than 0.6 is detected by UP. The right panel shows the ROC curve of Egr, criterion means that Egr is positive (suffering from echinococcosis) when detected in serum, and is negative (not suffering from echinococcosis) when greater than 0.99.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Example 1: process for identifying more hydatid protein by hydatid cyst separated after operation of hydatid patient

(1) Firstly, dividing the hydatid cysts separated after 10 hydatid patients into four parts to extract protein according to the tissue structure of the hydatid cysts, wherein the four parts are protocercaria, cyst fluid, a hair growth layer and a cornified layer from inside to outside; the 10 hydatid cysts were divided into active and inactive echinococcus cyst groups, depending on whether they were in the active phase or not.

(2) Extracting proteins from four components of each hydatid cyst, carrying out liquid enzymolysis on the extracted proteins, and then carrying out LC-MS/MS identification by using a QE mass spectrometer;

(3) Using maxquant protein identification software for original data of the QE machine, using a human and hydatid protein database collection to carry out hydatid protein identification, counting identification results and determining hydatid cyst parts with high hydatid protein content, wherein the results are shown in tables 1 and 2;

(4) As can be seen from tables 1 and 2, there are two basic conclusions: firstly, identifying more tissue parts of the hydatid protein as cyst fluid and metacercaria; and secondly, the proteins extracted from the cyst fluid and the metacercaria of the active hydatid cyst group contain more hydatid proteins.

TABLE 1 protein identification results of active hydatid cyst group

1 indicates metacercaria; 2 denotes cyst fluid; _3denotes the stratum corneum; and 4 denotes the cornified layer.

TABLE 2 identification of proteins from inactive hydatid cyst group

1 denotes miracidium; 2 denotes cyst fluid; _3denotes the stratum corneum; and 4 denotes the cornified layer.

Example 2: western Blot verification, gel cutting enzymolysis and mass spectrum identification (GeLC-MS/MS)

(1) Next, according to QE identification results of cyst fluid and metacercaria of 10 samples, we selected the metacercaria and cyst fluid components of five active echinococcus cyst groups of No.1-No.5 to respectively perform WB experiments with patient serum, so as to verify whether there is an immune reaction between the echinococcosis person and normal Tibetan human serum and the extracted holoprotein.

(2) As the total amount of protein extracted from the metacercaria components of a part of samples is very little, western Blotting experiment of cyst fluid component protein is mainly carried out, and the result is shown in figure 1, taking No.1 patient serum as an example, and showing the immunoreaction maps of echinococcosis person and normal Tibetan human serum and 5 cyst fluid component proteins. The immunoreactivity patterns of the sera of other patients and the sera of normal Tibetan patients to the 5 sample proteins were similar to those of the No.1 patient.

(3) According to the SDS-PAGE patterns of the 5 cyst fluid components, the SDS-PAGE gel is subjected to gel cutting enzymolysis by contrasting the immune bands of WB, so that more hydatid proteins in corresponding molecular weight sections can be identified through gel cutting separation, and the probability of finding hydatid antigens which have immunoreaction with serum is increased. In addition, this may reduce the impact of high abundance proteins on identification.

(4) In order to reduce the loss and the workload, 5 cyst fluid component proteins are uniformly divided into 10 components according to WB immune bands, and then each component is subjected to intracollagen enzymolysis.

(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 pool identification. The results are shown in Table 3.

(6) According to data analysis, the trypanosoma proteins identified by the gel cutting separation of the three samples of 1_2, 2 _2and 5 _2comprise the trypanosoma proteins identified by the rest four samples, so that candidates of subsequent trypanosoma antigens are searched from the trypanosoma proteins identified by the components of the three samples together.

TABLE 3 identification of proteins from five cyst fluid fractions by gel cutting and enzymolysis

The numbers in parentheses indicate the total identified protein, while the numbers outside the parenthesis indicate the hydatid protein in each fraction. And 2 denotes cyst fluid.

Example 3: screening of hydatid proteins as candidate antigens

(1) The common proteins identified by the same fraction in three samples served as candidate antigen pool, and finally a total of 93 unique proteins were produced by 10 fractions.

(2) These 93 proteins were first subjected to homology alignment with the human protein database, and after deleting the proteins with particularly high homology and those that have been selected as the hydatid antigen, the remaining proteins were subjected to prediction of B cell epitopes.

Example 4: epitope prediction and recombinant plasmid construction

(1) The Epitope Prediction uses an online Prediction tool http:// tools.iedb.org/bcll/, introduces protein sequences, selects a Bepipred Linear Epitope Prediction 2.0 Prediction method, and can obtain a sequence list about the protein which can be the Epitope.

(2) According to the antigen epitope list of each candidate protein, selecting sequence fragments containing one or more epitopes to form a sequence of the recombinant protein, introducing the sequence into an alignment tool to be aligned with a human protein database, and selecting the sequence fragments with the similarity of < =20% to the human protein as the recombinant protein.

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

Example 5: expression and purification of recombinant proteins

(1) The recombinant plasmid is transferred into BL21 (DE 3) for small-scale test expression, and after the fact that the protein can be expressed and the molecular weight is correct is confirmed, amplification culture is carried out.

(2) The collected bacterial liquid is subjected to ultrasonic protein extraction, and then nickel column purification is carried out through His-tag carried by plasmid, and FIG. 2 shows the purification result of the recombinant protein constructed by the UP fragment.

Example 6: western Blotting validation of UP recombinant proteins

(1) 8 of 14 recombinant proteins are successfully expressed, so that Western Blotting experiments are respectively carried out on the purified 8 recombinant proteins and 9 postoperative patient sera, the result shows that only 4 recombinant proteins have larger immunoreaction with the patient sera, wherein the UP recombinant proteins comprise the UP recombinant proteins, and the UP recombinant proteins are subsequently used as chafer antigen candidates to carry out ELISA verification tests on more patient sera.

(2) The experiment in figure 1 proves that the cyst fluid holoprotein has strong immunoreaction to the serum of a patient, but basically has no reaction to the serum of a normal Tibetan person, and the vast majority of protein bands causing immunoreaction are not high-abundance protein bands; FIG. 3 shows the WB experiment results of the UP recombinant protein and the commercial antigen Egr with the sera of 9 patients, the immunoreactivity of the UP recombinant protein is 56%, and that of the commercial antigen is 67% (only the samples with distinct bands between 43KD and 34KD are counted). 4 shows the B cell epitope, nucleotide sequence and amino acid sequence of the recombinant protein.

TABLE 4B-cell epitopes, nucleotide and amino acid sequences of UP recombinant proteins

Example 7: ELISA experiment of recombinant antigen UP recombinant protein

607 cases of B-ultrasonic positive plasma (containing suspected) and 636 cases of normal human plasma (determined to be negative) are detected, and the detection is carried out according to the standard operation steps of indirect ELISA, and the result shows that the positive detection rate of the UP recombinant protein is 90 percent, and the negative detection rate is 77 percent; and the positive detection rate and the negative detection rate of the same plasma were 86% and 92% respectively when the commercial antigen Egr (product name: echinococcus grandis, product number: YM-VI08, supplier: yiminou Biotech Co., ltd., hangzhou) was used for detection.

1. Specific operation steps of ELISA experiment

(1) Antigen quantification: each antigen was blown evenly using a pipette gun before coating and quantitated using a micro uv spectrophotometer to ensure that the protein was not degraded. No absorption peak at A280 indicates that the antigen amount is very low and is not suitable for coating; if the protein is separated out or insoluble, 8M urea is dripped into the mixture and then the mixture is evenly blown, centrifuged and the supernatant is taken for measuring the concentration.

(2) Coating: comparing the concentration of the antigen tube wall with the measured value, and then lowering the value; coating amount is 2 mug/ml and 10 ml/plate, and coating antigen amount is prepared according to actual use amount; labeling on coated ELISA plate: antigen number, name, label, coating date, plate number and the like, and if the coating concentration is not 2 mug/ml, the coating concentration needs to be indicated. After labeling the ELISA plates, coating antigen was added at 100. Mu.l/well, coated overnight at 4 ℃ or coated for 2h at 37 ℃.

(3) Washing the plate: the coated board is washed for 1 time by a board washing machine and then is patted dry on absorbent paper. The formula of the 50X lotion is as follows: 154.4g Tris, 149.0g NaCl, 20.0 ml Tween, 24.0ml pure water, and about 45ml concentrated hydrochloric acid for adjusting the pH value to 7.2, wherein the pure water has the capacity of 1000ml. Storing at 4 ℃.

(4) And (3) sealing: 2 percent of skimmed milk powder is used as a sealing solution for sealing, 200 mu l/hole is sealed overnight at 4 ℃ or sealed for 2h at 37 ℃.

(5) Washing the plate: the closed plate is washed 1 time with a plate washer and then patted dry on absorbent paper.

(6) Adding a primary antibody: the corresponding primary antibody was added using PBS in a ratio of 1: plasma was diluted 500 ℃ and incubated for 1h at 37 ℃.

(7) Washing the plate: washing the plate for more than 3 times by a plate washing machine, and patting the plate on absorbent paper.

(8) Adding secondary antibody (manufacturer: biyunyan; cargo number: A0201): since the primary antibody was derived from human plasma, the secondary antibody was administered using goat anti-human 1:500 additions were made and incubated at 37 ℃ for 1h.

(9) Washing the plate: and (5) washing the plate for more than 3 times by using a plate washing machine, and patting the plate on absorbent paper to dry. And preparing a color development liquid (TMB) while washing the plate.

(10) Color development: after the stop solution is prepared, 100 mul/hole of color developing solution is added, and the plate can be shaken to accelerate the color developing process and pay attention to close observation.

(11) TMB color development: when the color development reaches a certain degree (the blank and negative color development can not be too high generally), adding 50 mu l/hole stop solution, standing for more than 10min after adding the stop solution to ensure complete termination and uniform color (the plate can be shaken to accelerate the termination process), and reading after termination

(12) Reading: preheating the microplate reader for more than 30 min; the TMB color development detection wavelength is as follows: 450nm. And opening corresponding enzyme-labeling instrument measurement software, and reading. Store the data to the specified location and refine the data.

(13) Note that:

when the 96-well plate is loaded with a gun, care is taken to avoid air bubbles and to avoid wall hanging of the loaded sample.

After the sample is added, observing the whole plate, and requiring no bubbles at the bottom of the hole; when bubbles are found, the plate is shaken or the bubbles are removed by a gun head.

When the plate is washed, close attention is needed to ensure complete and thorough plate washing.

The color development needs to be closely paid attention, and the color development of negative, blank and positive controls is timely stopped.

(14) Various reagent formulations

Coating liquid: sodium carbonate-sodium bicarbonate buffer, ph9.6: na (Na) ₂ CO ₃ 1.59g，NaHCO ₃ 2.93g, and fixing the volume of pure water to 1000ml; and finally, detecting the pH value by using pH test paper. Storing at 4 deg.C.

2. Analysis of ELISA results

(1) And (3) negative and positive judgment standard: and selecting the average value (X) and Standard Deviation (SD) of the negative serum samples, wherein the upper limit cut-off value of the confidence interval is X +2SD. The OD value of the sample to be detected at 450nm is more than or equal to X +2SD, and the sample to be detected at less than X +2SD can be judged as positive, and the sample to be detected at less than X +2SD can be judged as negative.

(2) ROC curve: the optimal detection efficacy of the antigen was assessed using MedCalc software (version 19.4.0) to plot ROC curves (receptor Operating charateristic curve).

(3) By calculation, 607 cases of B-type positive plasma (containing suspected) and 636 cases of normal human plasma (determined as negative) are immunized by the hydatid antigen, the sensitivity is 90%, and the specificity is 77%; whereas the sensitivity was 86% and the specificity was 92% when the same plasma was tested with the commercial antigen. FIG. 4 shows ROC curves of UP recombinant protein and commercial antigen Egr and a boxplot for detecting OD values.

(4) Although the specificity of the UP recombinant protein is lower than that of the commercial antigen, the UP recombinant protein is more sensitive than the commercial antigen, and the dual-characteristic ROC curve of the UP recombinant antigen and the commercial antigen Egr is simulated through the logistic regression of MedCalc software, so that the detection efficiency of the two antigens together is very good. Therefore, the UP recombinant antigen can be used as a supplement detection of the commercial antigen to detect a sample which is not detected to be positive by the commercial antigen, and the UP recombinant protein can be used as a candidate of the neoantigen of the hydatid.

SEQUENCE LISTING

<110> Shenzhen Huashengshengsciences institute

<120> UP protein fragment and application thereof in human echinococcosis

<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 Gln Leu Thr Gln Arg

1 5 10

<210> 3

<211> 60

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 3

<400> 3

Val Pro Gly Gln Gly Asp Asn Met Pro Asp Leu Pro Ser Asp Trp Lys

1 5 10 15

Phe Pro Thr Met Gln Lys Ile Ser Glu Gly Ile Ser Glu Leu Cys Asp

20 25 30

Ser Leu Glu Leu Lys His Val Val Val Ile Gly Asp Gly Ala Gly Ala

35 40 45

Asn Ile 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 Ile 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 Ile 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 Gln Asn Phe Met Gln 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 Ile Arg Asn Arg Ser Ala Ser Met Asp Glu Tyr Asp Gln

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 Ile 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 Gln Lys Pro Gln Lys Ile 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 Gln Ser Leu Arg Glu Asn Ile Asn Pro Lys Asn Leu Asn

195 200 205

Lys Phe Ile Gln Ala Phe Met Val Arg Thr Asn Ile Thr Asp Ser Ile

210 215 220

Gly Asn Leu Lys Cys Pro Val Leu Phe Ile 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 Gln Pro Ala Arg Lys Ala Asn Val Glu Ile Leu Glu Ile Asp

260 265 270

Gly Val Ala Asn Val Met Arg Glu Arg Pro Glu Lys Val Ala Glu Ser

275 280 285

Val Gln Asn Phe Met Gln Gly Leu Gly Val Ala Ser Gly Ala Val Asn

290 295 300

Arg Arg Leu Ser Ala Thr Gly Asn Val Pro Lys Ile Arg Asn Arg Ser

305 310 315 320

Ala Ser Met Asp Glu Tyr Asp Gln Pro Val Gly Val Lys Asn Leu Ile

325 330 335

Tyr Asp Asn Thr Arg Arg Tyr Ser Lys Ala Thr Asp Ile Pro Glu Ile

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 (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, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9.

2. An isolated nucleic acid encoding the polypeptide of claim 1.

3. A recombinant expression vector comprising the isolated nucleic acid of claim 2.

4. A transformant comprising the isolated nucleic acid of claim 2 or the recombinant expression vector of claim 3, wherein the transformant is a bacterium or a eukaryotic cell.

5. The transformant according to claim 4, wherein the bacterium is E.

6. A kit for detecting echinococcosis, comprising the polypeptide of claim 1;

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 stopping solution and a diluting solution.

7. A marker combination comprising the polypeptide of claim 1; 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.

9. A method of producing the polypeptide of claim 1, comprising the steps of: culturing the transformant according to claim 4 under conditions suitable for fermentation thereof, so that the transformant expresses the polypeptide.

10. Use of a polypeptide according to claim 1 or a marker combination according to claim 7 for the preparation of an anti-echinococcosis antibody or a diagnostic agent for the diagnosis of diseases caused by echinococcosis granulosa.

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