CN114075270B

CN114075270B - Recombinant protein of human-derived echinococcosis antigen and application thereof

Info

Publication number: CN114075270B
Application number: CN202010825972.XA
Authority: CN
Inventors: 张聪敏; 任艳; 刘斯奇
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2020-08-17
Filing date: 2020-08-17
Publication date: 2024-03-26
Anticipated expiration: 2040-08-17
Also published as: CN114075270A

Abstract

The invention discloses a recombinant protein of a human echinococcosis antigen and application thereof. The recombinant protein comprises one or more of amino acid sequences shown as SEQ ID NO. 1-10; 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 retains the function of the recombinant protein. The immune response rate of the recombinant protein of the constructed human-derived echinococcosis antigen to a echinococcosis patient reaches 100%, and the recombinant protein can be used for preparing ELISA kits and clinically detecting human-derived echinococcosis. The single use or the combined use of the recombinant proteins can possibly improve the specificity and the sensitivity of laboratory diagnosis of the echinococcosis.

Description

Recombinant protein of human-derived echinococcosis antigen and application thereof

Technical Field

The invention belongs to the field of immunology, and particularly relates to a recombinant protein of a human echinococcosis antigen and application thereof.

Background

Echinococcosis, also known as echinococcosis, is a zoonosis. Human echinococcosis is mainly classified into cystic echinococcosis and bubble echinococcosis. Imaging is the most intuitive and most commonly used method for diagnosing echinococcosis, but serological detection is one of the important auxiliary means for imaging diagnosis of echinococcosis because the incubation period of echinococcosis is long and can be detected by imaging only when the capsule formed by echinococcosis is large to a certain extent. Currently, serological diagnostic antigens that can be used to diagnose echinococcosis are mainly Antigen B (anti-gen B) and Antigen 5 (anti-gen 5) and some recombinant antigens associated with both antigens.

To date, several methods for laboratory diagnosis of echinococcosis have been described, including detection of antibodies, antigens and cytokines. Among them, development of antibody detection mainly depends on development of the antigen species of the bag worm. However, both naturally purified antigens and recombinantly purified antigens have certain disadvantages in terms of specificity and/or sensitivity. More importantly, the human-derived echinococcosis causes such as difficult sampling, difficult separation of the echinococcosis proteins and the like, and no more antigens capable of being used for serological detection are presented.

The difficulties in developing antibodies against the artemia antigen in the prior art are as follows:

1) Extraction and identification of the bag worm protein from bag worm isolated after operation of the echinococcosis patient is a technical difficulty. If the identified artemia proteins are of a few kinds, it is more difficult to select proteins from which to choose a potential to be an artemia antigen.

2) The single antigen has few species. The most widely used diagnostic antigens for echinococcosis are antigen B and antigen 5, and many studies have shown that these two antigens produce certain false positives or false negatives when tested serologically.

3) Commercial artemia antigen is natural purified antigen, and soluble antigen fragments separated and purified from broken echinococcus granulosus are a mixture containing a plurality of artemia proteins. The more proteins that are mixed, the more likely they will produce more false negative or false positive results when tested serologically.

Therefore, there is a need for a diagnostic antigen capable of detecting echinococcosis with high sensitivity, high specificity and high immune response rate.

Disclosure of Invention

In order to solve the technical problems of lack of high-sensitivity, high-specificity and high-immunoreaction-rate echinococcosis detection diagnostic antigens in the prior art, the invention provides a recombinant protein of a human echinococcosis antigen and application thereof, in particular application in preparing anti-echinococcosis antibodies or diagnostic agents for diagnosing diseases caused by echinococcosis granulosa, and the recombinant protein has higher immunoreaction rate, specificity and sensitivity.

The first aspect of the present invention provides a recombinant protein 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, SEQ ID NO.9 and SEQ ID NO.10, or a mutation thereof;

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 retains the function of the recombinant protein.

Preferably, the recombinant protein comprises 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, SEQ ID NO.9 and SEQ ID NO. 10.

In a second aspect the invention provides an isolated nucleic acid encoding a recombinant protein according to the first aspect of the invention or a mutation thereof.

In a third aspect the invention provides a recombinant expression vector comprising an isolated nucleic acid according to the second aspect of the invention.

In a fourth aspect the present invention provides a transformant comprising an isolated nucleic acid according to the second aspect of the present invention or a recombinant expression vector according to the third 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 comprises the amino acid sequence shown as SEQ ID NO. 22.

In a fifth aspect the present invention provides a kit for the detection of echinococcosis comprising as antigen a recombinant protein according to the first aspect of the invention or a mutation thereof.

Preferably, the kit is an indirect ELISA detection kit, and the indirect ELISA detection kit further comprises a second antibody, a coating liquid, a washing liquid, a chromogenic liquid, a stop liquid and a diluent.

In a sixth aspect the invention provides an anti-echinococcosis siRNA or mRNA vaccine comprising an RNA sequence encoding a recombinant protein or a mutation thereof according to the first aspect of the invention.

A seventh aspect of the invention relates to the use of a recombinant protein according to the first aspect of the invention or a mutation thereof for the preparation of an anti-echinococcosis antibody.

An eighth aspect of the present invention relates to the use of a recombinant protein according to the first aspect of the present invention or a mutation thereof for the preparation of a diagnostic agent for the diagnosis of a disease caused by echinococcus granulosus.

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

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

The invention has the positive progress effects that:

1) The recombinant protein of the human-derived echinococcosis antigen is constructed, the immune response rate of the recombinant protein to a echinococcosis patient reaches 100 percent, and the recombinant protein can be used for preparing ELISA kits and clinically detecting human-derived echinococcosis.

2) The recombinant protein can be purified in a large quantity, and the human-derived echinococcosis can be conveniently screened on a large scale.

3) Since the recombinant protein has higher specificity and sensitivity against echinococcosis granulosa, the single use or the combined use of the recombinant protein can possibly improve the specificity and sensitivity of laboratory diagnosis of echinococcosis.

Drawings

FIG. 1 is a schematic diagram showing SDS-PAGE and Western blotting results of whole proteins extracted from seven cyst fluid fractions.

FIG. 2 is a schematic diagram showing the purification results of MG2 recombinant protein.

FIG. 3 is a schematic diagram showing the results of Western blotting verification experiments of the purified MG2 recombinant protein.

FIG. 4 is a box-shaped chart of ELISA results, wherein A is the ELISA results of MG2 and B is the ELISA results of Egr.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1

1. Identification of Baozhong protein by Baozhong capsule separated after operation of echinococcosis patient

1) Firstly, dividing the bag separated after the operation of 6 echinococcosis patients (first batch) into four parts according to the tissue structure thereof to extract proteins, wherein the four parts are primordium larvae, cyst fluid, germinal layers and horned cortex from inside to outside respectively; and the 6 bag worm sacs are divided into a bag liquid transparent group and a bag liquid non-transparent group according to the transparency of the bag liquid.

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

3) The original data of QE machine-down uses maxquat protein identification software, uses human and bag worm protein database collection to identify bag worm protein, counts identification result and determines bag worm bag part with more bag worm protein content, and the result is shown in table 1 and table 2;

4) As can be seen from table 1, there are two basic conclusions: firstly, more tissue parts of the identified bag worm protein are two parts of cyst fluid and metacercaria; and secondly, more bag worm proteins are contained in the protein extracted from the bag fluid and the metacercaria part of the bag fluid transparent group.

5) According to the basic conclusion of the first point, in order to identify more cyst proteins, the cyst fluid and the metacercaria of the bag of another 10 echinococcus patients (second batch) were additionally extracted, and as a result, as shown in tables 3 and 4, more cyst proteins were contained in the proteins extracted from the cyst fluid and metacercaria portions, which were still the transparent group of cyst fluids.

Table 1 protein identification of the clear group of cyst fluid in the first 6 echinococcosis group (1 represents primordium, 2 represents cyst fluid, 3 represents germinal layer, 4 represents horned cortex)

Table 2 protein identification results of the non-clear group of cyst fluid in the first 6 echinococcosis group (1 for primordium, 2 for cyst fluid, 3 for germinal layer, 4 for horny cortex)

Table 3 protein identification of the clear group of cyst fluid in the second group of 10 echinococcosis patients (1 for primordium, 2 for cyst fluid, 3 for germinal layer, 4 for horny cortex)

Table 4 protein identification results of the non-clear group of cyst fluid in the second group of 10 echinococcosis patients (1 for primordium, 2 for cyst fluid, 3 for germinal layer, 4 for horny cortex)

2. Western blotting verification, gel cutting enzymolysis and mass spectrometry identification (GeLC-MS/MS)

1) According to QE identification results of the cyst fluid and the metacercaria of 16 samples, seven cyst fluid transparent group samples of No.1, no.2, no.3, no.7, no.8, no.9 and No.10 are selected, and Western blotting experiments are respectively carried out on metacercaria and cyst fluid components of the cyst fluid transparent group samples and blood plasma of patients so as to verify whether immune reaction exists between blood plasma of echinococcosis patients and normal persons and the extracted whole protein.

2) Because the total amount of protein extracted from the original cercaria component of a part of samples is small, western blotting experiments of cyst fluid component proteins of cyst fluid transparent samples are mainly carried out, and the results are shown in figure 1, and taking No.1 patient plasma as an example, the immune response patterns of the cyst fluid component proteins of the cyst fluid transparent samples of the echinococcosis patient, normal human plasma and seven cyst fluid transparent groups are shown. The immune response patterns of the plasma of other patients and the plasma of normal people on seven sample proteins are similar to those of the No.1 patient.

The results show that the total protein extracted from the seven cyst fluid components has a strong immune response to the plasma of patients, but has no substantial response to the plasma of normal people.

3) According to the SDS-PAGE profile of 2), we performed gel cutting and enzymolysis on SDS-PAGE gel against Western blotting immune bands, so as to identify more artemia proteins in the corresponding molecular weight section by gel cutting and separation, and increase the probability of finding artemia antigens immunoreactive with blood plasma. In addition, this reduces the impact of high abundance proteins on the identification.

4) In order to reduce loss and reduce workload, the cyst fluid component proteins in the seven cyst fluid transparent component samples are uniformly divided into 10 components according to Western blotting immune bands, and then the components are subjected to intra-gel enzymolysis respectively.

5) After LC-MS/MS analysis by QE-HF-X mass spectrometer, the maxquat software performed protein search identification using the same database as in the identified proteins. The results are shown in Table 5, with the numbers in brackets indicating the total identified protein, and the numbers outside the brackets indicating the inclusion of a worm protein in each component.

6) From the data analysis, the three samples 1_2, 2_2 and 10_2 are cut and separated to obtain the identified bag worm protein which contains bag worm proteins identified by the other four samples, so that the candidate of the follow-up bag worm antigen is searched from the bag worm proteins commonly identified by all components of the three samples.

TABLE 5 identification results of protein fragments of capsular component protein-cutting enzymolysis in seven capsular liquid transparent samples

3. Screening for Baotong proteins as candidate antigens

1) Using the common proteins identified in the three samples of the same component as candidate antigen libraries, a total of 93 proteins were produced from the final 10 components.

2) These 93 proteins were first aligned for homology to the human protein database, and after deletion of proteins or protein fragments with more than 20% homology, and those proteins that had been selected as the artemia antigen, the remaining proteins were predicted for B cell epitopes.

4. Epitope prediction and recombinant plasmid construction

1) The sequence list of the possible antigen epitope of the protein, namely the antigen epitope list, can be obtained by introducing the protein sequence by using an online prediction tool http:// tools.

2) According to the 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 only sequence fragments with similarity < = 20% with human homologous proteins are selected to construct the recombinant protein.

3) Finally, 11 protein sequence fragments were obtained and inserted into pET-30a (+) plasmid by molecular cloning technique to construct recombinant plasmid, and the fragments were confirmed to have been inserted by sequencing.

5. Expression and purification of recombinant proteins

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

2) And (3) carrying out ultrasonic extraction on the recombinant protein from the collected bacterial liquid, and then carrying out nickel column purification through His-tag carried by plasmids.

6. Western blotting verification of recombinant plasmid expression protein

The purified 11 recombinant proteins and commercial antigens (the cabbage caterpillar purified antigen Echinococcus granulosus, egr, yiminox biotechnology Co., hangzhou, YM-VI 08) are respectively subjected to Western blotting experiments with 16 post-operation patient plasma samples, and the result shows that the immune response rate of only 4 recombinant proteins and 16 post-operation patient plasma samples is greater than or equal to the commercial antigens, so that the 4 recombinant proteins are selected as candidate cabbage caterpillar antigens for ELISA verification experiments of more plasma samples.

In this example, a recombinant plasmid was constructed using a truncated protein of Murinoglobulin-2 protein (MG 2 recombinant protein for short) as an example.

As shown in Table 6, the MG2 recombinant protein comprises 10 antigen epitopes, the amino acid sequence of the antigen epitopes is shown as SEQ ID NO. 1-10, and the nucleotide sequence is shown as SEQ ID NO. 11-20.

The recombinant plasmid comprises an MG2 recombinant protein nucleotide sequence shown as SEQ ID NO. 21, and the corresponding MG2 recombinant protein amino acid sequence is shown as SEQ ID NO. 22.

The purification result of the MG2 recombinant protein is shown in FIG. 2, wherein the gradient elution result of the MG2 recombinant protein is shown in the figure, and the loading amount of each lane is 10 mu l.

The Western blotting verification result of the MG2 recombinant protein is shown in figure 3, and when only a sample with obvious bands between 43KD and 34KD is counted, the immunoreaction rate of the MG2 recombinant protein is 100%, and the immunoreaction rate of commercial antigen is 75%.

In the case of using commercial Antigen Egr, the two antigens, anti-gen B and anti-gen 5, are mainly relied on, wherein the anti-gen B has the most important molecular weight between 43KD and 34KD in diagnosing human echinococcosis, and immune reaction occurs at 16KD and 24KD, etc. due to the action of anti-gen B on plasma of patients after degradation into protein subunits, as also demonstrated by Western blotting results shown in FIG. 3.

Unlike the method of directly using DNA of the bag worm tissue to perform PCR amplification to obtain the sequence related to the bag worm antigen based on the forward thinking of genomics, the bag worm antigen is obtained based on the reverse thinking of proteomics in the embodiment, and is screened out by gel cutting enzymolysis and mass spectrometry detection, namely GeLS-MS/MS technology. Essentially, the target is obtained by proteomics technology, and a certain experimental verification basis is provided.

Table 6B cell epitope, nucleotide sequence and amino acid sequence of MG2 recombinant protein

Example 2

In this example, the MG2 recombinant protein described in example 1 was used, and the test subjects were 60B-ultrasonic positive plasma (suspected) samples and 33 normal human plasma (negative) samples, and the test was performed according to the standard procedure of the indirect ELISA.

1. ELISA Experimental procedure

1) Antigen quantification: each antigen was homogenized using a pipette before coating and quantified using a micro-uv spectrophotometer to ensure that the protein was not degraded. The absence of an absorption peak at A280 indicates that the amount of antigen is low and is not suitable for coating; if the protein is precipitated or insoluble, 8M urea is added dropwise and then blown up, the supernatant is centrifuged and the protein concentration is re-quantified using the Bradford method.

2) Coating: comparing the concentration of the antigen tube wall with the measured value, and taking the value low; the coating amount is 2 mug/ml, 10 ml/plate, and the antigen coating amount is prepared according to the actual use amount;

coated ELISA plates were labeled: antigen number, name, label, coating date, plate number, etc., and if the coating concentration is not 2. Mu.g/ml, the coating concentration needs to be indicated. After the ELISA plates were labeled, 100. Mu.l/well of coating antigen was added, and the coating was carried out at 4℃overnight or at 37℃for 2 hours.

3) Washing the plate: the coated board is washed for 1 time by a board washing machine, and is beaten to be dry on absorbent paper.

4) Closing: 200 μl/well of 2% nonfat dry milk was blocked as blocking solution, overnight at 4deg.C or for 2h at 37deg.C.

5) Washing the plate: the sealed board is washed 1 time by a board washing machine and is beaten to be dry on the absorbent paper.

6) Adding an antibody: plasma samples were added and the plasma diluted 1:500 by volume using PBS as diluent and incubated for 1h at 37 ℃.

7) Washing the plate: washing the board with a board washing machine for 5 times, and drying the board by beating on absorbent paper.

8) Adding a secondary antibody (secondary antibody): since the primary antibody was derived from human plasma, goat anti-human secondary antibody (Biyun Tian, A0201) was added at a volume ratio of 1:500 and incubated at 37℃for 1h.

9) Washing the plate: washing the board with a board washing machine for 5 times, and drying the board by beating on absorbent paper. And washing the plate, and preparing a color development liquid TMB.

10 Color development): after preparing the stop solution (2M sulfuric acid), 100. Mu.l/Kong Xianse solution was added, and the plate was allowed to shake to accelerate the development process and observe closely.

11 TMB color development): when the color is developed for 4min (blank and negative color cannot be developed too high generally), 100 mu l/hole of stop solution is added, and the mixture is kept stand for 10min after the stop solution is added, so that the stop is thorough and the color is uniform (the plate can be rocked to accelerate the stop process), and the reading is carried out after the stop.

12 Reading: the enzyme label instrument must be preheated for more than 30 minutes; the TMB chromogenic detection wavelength was 450nm. Opening corresponding enzyme label instrument measuring software to read; store the data to the specified location and refine the data.

13 Notice of:

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

After sample addition, observing the whole plate, wherein no bubble is required at the bottom of the hole; when the bubbles are found, the plate is shaken or the pipette tip is used to remove the bubbles.

The close attention is needed when the plate is washed, so that the plate is completely and thoroughly washed.

The color development needs to be closely concerned, and the color development conditions of negative, blank and positive controls are combined to be terminated in time.

14 Various reagent formulations:

the coating uses a coating liquid, which comprises: pH value9.6 sodium carbonate-sodium bicarbonate buffer, 1.59gNa ₂ CO ₃ 、2.93g NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the The volume was set to 1000ml using pure water, the pH was checked with pH paper and stored at 4 ℃.

The wash plate uses a 50X wash solution comprising: 154.4g Tris, 149.0g NaCl, 24.0ml Tween-20 and 800ml pure water; the pH was adjusted to 7.2 using about 45ml of concentrated hydrochloric acid, purified water was set to 1000ml and stored at 4 ℃.

2. ELISA result analysis

1) Negative and positive judgment criteria: the mean (X) and Standard Deviation (SD) of negative plasma samples were selected with the upper confidence interval cut-off value being X+2SD. The OD value of the sample to be tested at 450nm is greater than or equal to X+2SD, and can be judged as positive, and less than X+2SD can be judged as negative.

2) By calculation, as shown in fig. 4, the left panel a shows the ELISA results of MG2 recombinant protein, the right panel B shows the ELISA results of Egr, where mg2_od (+) and egr_od (+) refer to the plasma OD values determined to be positive by MG2 or Egr detection, and mg2_od (-) and egr_od (-) refer to the plasma OD values determined to be negative by MG2 or Egr detection. The MG2 recombinant protein of this example was used as a bag worm antigen to immunize 60 cases of B-ultrasonic positive plasma (containing suspected) and 33 cases of normal human plasma (determined as negative), with a positive detection rate of 80% and a negative detection rate of 100%; the positive detection rate is 86% and the negative detection rate is 100% when commercial antigens are used for detecting the same plasma, so that the sensitivity of the MG2 recombinant protein in the embodiment reaches 80% and the specificity is 100%.

3) Although the positive detection rate of the MG2 recombinant protein is lower than that of the commercial antigen, the MG2 recombinant protein detects the positive of the sample which is not detected to be positive by the commercial antigen, and therefore the MG2 recombinant protein can be used as a supplementary antigen for clinically detecting the human echinococcosis by using the commercial antigen. According to Western blotting results, the immunoreaction rate of the MG2 recombinant protein to 16 cases of postoperative echinococcosis human plasma is 100%.

SEQUENCE LISTING

<110> Shenzhen Hua big Gene Co., ltd

<120> a recombinant protein of human-derived echinococcosis antigen and use thereof

<130> P20013554C

<160> 22

<170> PatentIn version 3.5

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<211> 8

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<213> Artificial Sequence

<220>

<223> epitope 1

<400> 1

Glu Ala Thr Gly Ser Ser Ile Val

1 5

<210> 2

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 2

<400> 2

Arg Met Trp Pro Glu Pro Lys Leu Glu Leu Lys

1 5 10

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 3

<400> 3

Val Ser Asn Glu Arg Glu Glu

1 5

<210> 4

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 4

<400> 4

Val Thr Asp Pro Cys Gly

1 5

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 4

<400> 5

Ala Asp Asn Asn Pro Thr Arg

1 5

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 6

<400> 6

Ser Val Thr Pro Gly Lys Glu Ala His Asn Phe

1 5 10

<210> 7

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 7

<400> 7

Ser Asn Cys Thr Asp Glu Asn Ser Ser Pro Lys Ile Asn

1 5 10

<210> 8

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 8

<400> 8

Gly Asn Arg Leu Ala Ser

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 9

<400> 9

Cys Lys Gly Val Ser Lys Gly

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> epitope 10

<400> 10

Tyr Ile Gln Pro Gly Ala Val Asn Val

1 5

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 1

<400> 11

gaagctaccg gttcctccat tgtg 24

<210> 12

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 2

<400> 12

cgaatgtggc ccgagcccaa actagagctg aag 33

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 3

<400> 13

gtctcgaatg aaagagaaga a 21

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 4

<400> 14

gtaactgatc cttgcggt 18

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 5

<400> 15

gctgataata atccgactcg a 21

<210> 16

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 6

<400> 16

tccgtcacac caggaaaaga agcacacaat ttt 33

<210> 17

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 7

<400> 17

agtaattgca ctgacgagaa ttcatcacca aaaattaat 39

<210> 18

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 8

<400> 18

ggcaaccgtc tagcctca 18

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 9

<400> 19

tgcaagggcg tttctaaagg t 21

<210> 20

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> epitope 10

<400> 20

tacatccagc caggtgcagt gaatgtc 27

<210> 21

<211> 711

<212> DNA

<213> Artificial Sequence

<220>

<223> MG2 recombinant protein

<400> 21

gaagctaccg gttcctccat tgtggtctct ggcatcacta ccttccgaat gtggcccgag 60

cccaaactag agctgaagat tcccagttcg tttaggtacg gtatcccaat cgctggtcag 120

gtgctataca gaagcgtctc gaatgaaaga gaagaattgg aagtcattgt aagggaggta 180

actgatcctt gcggtggatg gatgatgctc gctgataata atccgactcg attgaagcga 240

atcatctccg tcacaccagg aaaagaagca cacaattttg tgttgccacc gctaaatttc 300

aagaatagcg cttcagtact ggtgcgacgg atttgcaaga gtaattgcac tgacgagaat 360

tcatcaccaa aaattaatca tcgctatttc ggattgctgt actcctttga ttatggcaac 420

cgtctagcct catggaatac cacaatacat gtgttggaga gctgtgaatg caagggcgtt 480

tctaaaggtg cttttggcct cctcggttcg tacatccagc caggtgcagt gaatgtcgat 540

ctcgcagagc ttgaggtagt gacgcttaag ggtatcgtca aagtagcccc actgttcaaa 600

gagatgctgg agatgaagat gaaggaaatg agagagaatg agcaggagtt atggtgtgcc 660

agtagaaagc ccttctttgt tcttctccag aagcttacga gtcagcagat a 711

<210> 22

<211> 237

<212> PRT

<213> Artificial Sequence

<220>

<223> MG2 recombinant protein

<400> 22

Glu Ala Thr Gly Ser Ser Ile Val Val Ser Gly Ile Thr Thr Phe Arg

1 5 10 15

Met Trp Pro Glu Pro Lys Leu Glu Leu Lys Ile Pro Ser Ser Phe Arg

20 25 30

Tyr Gly Ile Pro Ile Ala Gly Gln Val Leu Tyr Arg Ser Val Ser Asn

35 40 45

Glu Arg Glu Glu Leu Glu Val Ile Val Arg Glu Val Thr Asp Pro Cys

50 55 60

Gly Gly Trp Met Met Leu Ala Asp Asn Asn Pro Thr Arg Leu Lys Arg

65 70 75 80

Ile Ile Ser Val Thr Pro Gly Lys Glu Ala His Asn Phe Val Leu Pro

85 90 95

Pro Leu Asn Phe Lys Asn Ser Ala Ser Val Leu Val Arg Arg Ile Cys

100 105 110

Lys Ser Asn Cys Thr Asp Glu Asn Ser Ser Pro Lys Ile Asn His Arg

115 120 125

Tyr Phe Gly Leu Leu Tyr Ser Phe Asp Tyr Gly Asn Arg Leu Ala Ser

130 135 140

Trp Asn Thr Thr Ile His Val Leu Glu Ser Cys Glu Cys Lys Gly Val

145 150 155 160

Ser Lys Gly Ala Phe Gly Leu Leu Gly Ser Tyr Ile Gln Pro Gly Ala

165 170 175

Val Asn Val Asp Leu Ala Glu Leu Glu Val Val Thr Leu Lys Gly Ile

180 185 190

Val Lys Val Ala Pro Leu Phe Lys Glu Met Leu Glu Met Lys Met Lys

195 200 205

Glu Met Arg Glu Asn Glu Gln Glu Leu Trp Cys Ala Ser Arg Lys Pro

210 215 220

Phe Phe Val Leu Leu Gln Lys Leu Thr Ser Gln Gln Ile

225 230 235

Claims

1. A transformant comprising an isolated nucleic acid encoding a recombinant protein having the amino acid sequence shown in SEQ ID No. 22, said transformant being a bacterial or eukaryotic cell.

2. The transformant according to claim 1, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO. 21.

3. The transformant of claim 1 or 2, wherein the bacterium is e.coli bl21.

4. A kit for detecting echinococcosis is characterized by comprising recombinant proteins with amino acid sequences shown as SEQ ID NO. 22.

5. The kit of claim 4, wherein the kit is an indirect ELISA detection kit.

6. The kit of claim 4 or 5, wherein the indirect ELISA assay kit further comprises a secondary antibody, coating solution, wash solution, color development solution, stop solution, and dilution solution.

7. The application of the recombinant protein with the amino acid sequence shown as SEQ ID NO. 22 in preparing a diagnostic agent for diagnosing diseases caused by echinococcus granulosus.