A protein fragment of psap-3 and its application in human hydatidosis

Abstract

The application discloses a PSAP-3 protein fragment and application thereof in human echinococcosis. The polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The application further discloses a recombinant protein or a mutation thereof, wherein the recombinant protein comprises an amino acid sequence as shown in SEQ ID NO:5, and the amino acid sequence of the mutation has at least 80% identity with the amino acid sequence of the recombinant protein. The immunoreaction rate of the PSAP-3 recombinant protein of the application is 100%, the echinococcosis can be effectively detected, the positive detection rate is 96%, the negative detection rate is 90%, and the positive patient that cannot be detected by Egr can be detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, and in particular to a PSAP-3 protein fragment, a recombinant protein or a mutation thereof, a nucleic acid sequence and application thereof in detecting or treating human hydatid disease. BACKGROUND

[0002] Hydatid disease, also known as echinococcosis, is a zoonosis. Human hydatid disease is mainly divided into cystic hydatid disease and alveolar hydatid disease. Imaging detection is the most intuitive and most commonly used method for diagnosing hydatid disease, but since the incubation period of hydatid disease is long, and only when the hydatid cyst formed by hydatid disease reaches a certain size can it be detected by imaging, serological detection is one of the important auxiliary means for imaging diagnosis of hydatid disease. At present, the serological diagnostic antigens that can be used to diagnose hydatid disease mainly include antigen B (Antigen B) and antigen 5 (Antigen 5) and some recombinant antigens related to the two antigens.

[0003] It is itself a technical difficulty to extract and identify hydatid proteins from hydatid cysts isolated from postoperative hydatid patients. It will be more difficult to select proteins that may serve as hydatid antigens if the types of hydatid proteins identified are few.

[0004] Secondly, the single antigen is few in type. The most widely used diagnostic antigen for hydatid disease is antigen B and antigen 5, and many studies have shown that these two antigens will produce certain false positives or false negatives in serum detection.

[0005] Commercial hydatid antigens are natural purified antigens, which are isolated and purified from soluble antigen fragments after crushing of fine-grained echinococcosis, and are a mixture containing multiple hydatid proteins. The more mixed proteins, the more likely it is to produce more false negatives or false positives in serum detection. SUMMARY

[0006] To solve the technical defects of the prior art such as difficulty in extracting and identifying hydatid proteins and few hydatid antigens, the present application provides a PSAP-3 protein fragment and its application in human hydatid disease. By finding more antigens that can be used to diagnose hydatid disease, the present application expands the hydatid antigen database, so that these antigens, either alone or in combination, can improve the specificity and sensitivity of laboratory diagnosis of hydatid disease. In addition, a mature experimental process for extracting and identifying more hydatid proteins from hydatid cysts isolated from postoperative hydatid patients is also produced.

[0007] To solve the above technical problems, one of the schemes of the present application provides a polypeptide, wherein the polypeptide comprises one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] To solve the above technical problems, the sixth aspect of the present application provides a recombinant protein or a mutation thereof, wherein the recombinant protein comprises an amino acid sequence as shown in SEQ ID NO: 5; the amino acid sequence of the mutation has at least 80%, 85%, 90%, 95%, 98%, 99% identity with the amino acid sequence of the recombinant protein, and maintains the function of the recombinant protein.

[0009] To solve the above technical problems, the third aspect of the present application provides an isolated nucleic acid, wherein the isolated nucleic acid encodes the polypeptide according to the first aspect of the present application or the recombinant protein or the mutation thereof according to the second aspect of the present application; preferably, the nucleotide sequence encoding the recombinant protein according to the second aspect of the present application is shown in SEQ ID NO: 6.

[0010] To solve the above technical problems, the fourth aspect of the present application provides a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid according to the third aspect of the present application.

[0011] To solve the above technical problems, the fifth aspect of the present application provides a transformant, wherein it comprises the isolated nucleic acid according to the third aspect of the present application or the recombinant expression vector according to the fourth aspect of the present application, and wherein the transformant is a bacterium or a eukaryotic cell.

[0012] Preferably, the bacterium is E. coli BL21.

[0013] More preferably, the transformant expresses an amino acid sequence as shown in SEQ ID NO: 1-5.

[0014] To solve the above technical problems, the sixth aspect of the present application provides a kit for detecting hydatid disease, wherein the kit comprises the polypeptide according to the first aspect of the present application or the recombinant protein or the mutation thereof according to the second aspect of the present application.

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

[0016] More preferably, the indirect ELISA detection kit further comprises a secondary antibody, a coating solution, a washing solution, a color developing solution, a termination solution, and a dilution solution.

[0017] To solve the above technical problems, the seventh aspect of the present application provides a marker combination, wherein the combination comprises the polypeptide according to the first aspect of the present application or the recombinant protein or the mutation thereof according to the second aspect of the present application.

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

[0019] To solve the above technical problems, the eighth aspect of the present application provides an anti-echinococcosis siRNA or mRNA vaccine, wherein the siRNA or mRNA vaccine comprises an RNA sequence complementary to a nucleotide sequence encoding the polypeptide according to any one of the first to seventh aspects of the present application or the recombinant protein or mutation thereof according to the second aspect of the present application.

[0020] To solve the above technical problems, the ninth aspect of the present application provides a method for producing the polypeptide according to any one of the first to seventh aspects of the present application or the recombinant protein or mutation thereof according to the second aspect of the present application, wherein the method comprises the following steps: culturing the transformant according to the fifth aspect of the present application under conditions suitable for fermentation thereof to allow the polypeptide or the recombinant protein or mutation thereof to be expressed.

[0021] To solve the above technical problems, the tenth aspect of the present application provides a use of the polypeptide according to any one of the first to seventh aspects of the present application or the recombinant protein or mutation thereof according to the second aspect of the present application or the marker combination according to the seventh aspect of the present application in the preparation of an anti-echinococcosis antibody or a diagnostic agent for diagnosing a disease caused by echinococcosis granulosus.

[0022] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present application.

[0023] The reagents and raw materials used in the present application are commercially available.

[0024] The positive progress effect of the present application is that:

[0025] 1) A new echinococcosis antigen candidate PSAP-3 is screened out, which can be used to prepare an ELISA kit and for clinical detection of human echinococcosis, and can be used as a mutual supplement with Egr antigen.

[0026] 2) The echinococcosis new antigen can be purified in large quantities, which is convenient for large-scale screening of human echinococcosis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 SDS-PAGE and Western blotting results of total proteins extracted from five cyst fluid components are shown. The left graph shows the WB results of using No. 1 patient serum as primary antibody and five cyst fluid components as antigen. The middle graph shows the WB results of using normal Tibetan serum as primary antibody and five cyst fluid components as antigen. The right graph shows the protein gel results of five cyst fluid components. The experiment proves that the total protein of the cyst fluid has a strong immune response to the patient's serum, and has basically no response to the normal Tibetan serum, and most of the protein bands causing immune response are not high-abundance protein bands.

[0028] Figure 2The results of the purification of PSAP-3 recombinant protein are shown in the figure. The gradient elution results of PSAP-3 recombinant protein are shown in the figure, and the amount of each lane is 10 μl. Each lane is respectively referred to as the whole protein of the bacteria after ultrasonic, the flow through after loading on the nickel column, and the elution gradient from 20 mM imidazole to 1 M imidazole;

[0029] Figure 3 The results of the WB verification experiment of the purified PSAP-3 recombinant protein are shown. Numbers 1-9 refer to 9 patient sera taken before hydatid cyst removal surgery. Control refers to normal Tibetan serum without disease. Egr_SDS-PAGE refers to the protein gel results of the commercial antigen Egr, and the protein band indicated by the arrow is Antigen B;

[0030] Figure 4 The results of the ELISA experiment are shown. PSAP-3_OD(+) and Egr_OD(+) refer to the OD values of 607 B-ultrasound positive plasma, and PSAP-3_OD(-) and Egr_OD(-) refer to the OD values of 636 normal human plasma.

[0031] Figure 5 The ROC curves of PSAP-3 recombinant protein and commercial antigen Egr are shown. The left figure is the ROC curve of PSAP-3, and Criterion refers to the detection of serum by PSAP-3, greater than 0.64 is positive (suffering from echinococcosis), and less than or equal to 0.64 is negative (not suffering from echinococcosis). The right figure is the ROC curve of Egr, and Criterion refers to the detection of serum by Egr, greater than 0.99 is positive (suffering from echinococcosis), and less than or equal to 0.99 is negative (not suffering from echinococcosis). DETAILED DESCRIPTION

[0032] The present application will be further described in the following examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the instructions of the commercial product.

[0033] Example 1: Process of identifying more echinococcus proteins from hydatid cysts isolated after surgery of echinococcosis patients

[0034] (1) First, 10 hydatid cysts isolated after surgery of echinococcosis patients were divided into four parts according to their tissue structure to extract proteins, which were scolex, cyst fluid, germinal layer and cuticle layer from inside to outside; and 10 hydatid cysts were divided into active hydatid cyst group and non-active hydatid cyst group according to whether they were in active period.

[0035] (2) Protein extraction was performed on each of the four components of the hydatid cyst, and the extracted protein was subjected to liquid enzymatic hydrolysis, followed by LC-MS / MS identification using QE mass spectrometer;

[0036] (3) The raw data of QE were used to identify the proteins of the cysticerci by using the maxquant software and the database of human and cysticerci proteins. The identification results were counted and the cysticerci protein content of the cysticerci was determined. The results are shown in Tables 1 and 2.

[0037] (4) From Tables 1 and 2, two basic conclusions can be drawn: first, the parts of the cysticerci with more identified cysticerci proteins are the hydatid fluid and the protoscolex; second, only the hydatid fluid and the protoscolex of the active cysticerci group contain more cysticerci proteins.

[0038] Table 1 Protein identification results of the active cysticerci group

[0039]

[0040] _1 represents the protoscolex; _2 represents the hydatid fluid; _3 represents the germinal layer; and _4 represents the cuticular layer.

[0041] Table 2 Protein identification results of the non-active cysticerci group

[0042]

[0043] _1 represents the protoscolex; _2 represents the hydatid fluid; _3 represents the germinal layer; and _4 represents the cuticular layer.

[0044] Example 2: Western Blot verification, gel digestion and mass spectrometry identification (GeLC-MS / MS)

[0045] (1) Next, according to the QE identification results of the hydatid fluid and the protoscolex of the 10 samples, we selected the protoscolex and the hydatid fluid components of the five active cysticerci groups of No. 1-No. 5 to perform WB experiments with the patient serum, in order to verify whether there is an immune response between the cysticercosis patient and the normal Tibetan serum and the extracted total protein.

[0046] (2) Because the total amount of protein extracted from the protoscolex component of part of the samples is very small, we mainly performed Western Blotting experiments on the hydatid fluid component protein. The results are shown in Figure 1 , which shows the immune response map of the cysticercosis patient and the normal Tibetan serum with the five hydatid fluid component proteins. The immune response maps of other patient serum and normal Tibetan serum with the five sample proteins are similar to those of No. 1 patient.

[0047] (3) According to the SDS-PAGE patterns of the five cystic fluid components, we cut the SDS-PAGE gel for in-gel digestion to identify more proteins in the corresponding molecular weight segments, in order to increase the probability of finding cysticercus antigens that can react with serum. In addition, this can reduce the impact of high-abundance proteins on identification.

[0048] (4) In order to reduce losses and workload, we divided the five cystic fluid components into 10 components according to the WB immunobands, and then each component was subjected to in-gel digestion.

[0049] (5) After LC-MS / MS analysis by QE-HF-X mass spectrometer, maxquant software used the same database as in 2.2.1 for protein library identification. The results are shown in Table 3.

[0050] (6) Through data analysis, it is known that the cysticercus proteins identified by in-gel separation of 1_2, 2_2 and 5_2 samples contain the cysticercus proteins identified by the other four samples, so the subsequent candidate cysticercus antigens are searched from the cysticercus proteins identified from the three samples.

[0051] Table 3 Protein identification results of in-gel digestion of five cystic fluid components

[0052]

[0053] The numbers in parentheses represent the total number of proteins identified, and the numbers outside the parentheses represent the cysticercus proteins in each component. _2 indicates cystic fluid.

[0054] Example 3: Screening of cysticercus proteins as candidate antigens

[0055] (1) The common proteins identified in the same component in three samples were used as the candidate antigen library, and finally 10 components produced a total of 93 unique proteins.

[0056] (2) These 93 proteins were first subjected to homology comparison with human protein database, and after deleting proteins with high homology and those already selected as cysticercus antigens, the remaining proteins were subjected to prediction of B cell antigen epitopes.

[0057] Example 4: Antigen epitope prediction and construction of recombinant plasmid

[0058] (1) Antigen epitope prediction used an online prediction tool http: / / tools.iedb.org / bcell / , imported the protein sequence, and selected Bepipred Linear Epitope Prediction 2.0 prediction method to obtain a list of sequences that may become antigen epitopes of the protein.

[0059] (2) We select the sequence fragments containing one or more epitopes according to the antigen epitope list of each candidate protein to form the sequence of the recombinant protein, and introduce the sequence into the alignment tool for alignment with the human protein database. Only the sequence fragments with a similarity of <= 20% to human proteins are selected to construct the recombinant protein.

[0060] (3) Finally, 14 protein sequence fragments are inserted into pET-30a(+) plasmids by molecular cloning technology to construct recombinant plasmids, and the fragments are confirmed to have been inserted by sequencing.

[0061] Example 5: Expression and purification of recombinant protein

[0062] (1) The recombinant plasmid is transformed into BL21(DE3) for small-scale expression, and after confirming that the protein can be expressed and the molecular weight is correct, the culture is expanded.

[0063] (2) The collected bacterial solution is subjected to ultrasonic extraction of protein, and then subjected to nickel column purification by His-tag carried by the plasmid, Figure 2 The purification results of the recombinant protein constructed by the PSAP-3 fragment of the application are shown.

[0064] Example 6: Western Blotting verification of PSAP-3 recombinant protein

[0065] (1) Among the 14 recombinant proteins, 8 are successfully expressed, so the 8 recombinant proteins after purification are subjected to Western Blotting experiments with 9 postoperative patient sera, and the results show that only 4 recombinant proteins have a larger immune response with the patient serum, which contains the PSAP-3 recombinant protein. The PSAP-3 recombinant protein is used as a candidate for more patient serum ELISA verification experiments.

[0066] (2) Figure 3 The WB experiment results of PSAP-3 recombinant protein and commercial antigen Egr (product name: Echinococcus granulosus purified antigen, product number: YM-VI08, supplier: Hangzhou Yiminuo Biological Technology Co., Ltd.) with 9 patient sera are shown, and the results show that the immune response rate of PSAP-3 recombinant protein is 100%, and the immune response rate of commercial antigen is 67% (only samples with obvious bands between 43KD and 34KD are counted). PSAP-3 recombinant protein can detect positive patients 1, 2 and 7, while Egr cannot. Table 4 shows the B cell antigen epitope, nucleotide sequence and amino acid sequence of the recombinant protein.

[0067] Table 4 B cell epitope, nucleotide sequence and amino acid sequence of PSAP-3 recombinant protein

[0068]

[0069] Example 7: ELISA experiment of recombinant antigen PSAP-3 recombinant protein

[0070] The detected subjects were 607 cases of B-ultrasound positive plasma (including suspected) and 636 cases of normal human plasma (determined as negative), which were implemented according to the standard operation procedure of indirect ELISA. The results showed that the positive detection rate of PSAP-3 recombinant protein was 96%, and the negative detection rate was 90%; while the positive detection rate of the same plasma using commercial antigen Egr was 86%, and the negative detection rate was 92%.

[0071] 1. Specific operation steps of ELISA experiment

[0072] (1) Antigen quantification: Each antigen was blown evenly before coating using a pipette, and the antigen was quantified using a micro UV spectrophotometer to ensure that the protein was not degraded. There was no absorption peak at A280, indicating that the antigen amount was too low to be used for coating; if the protein precipitated or was not soluble, 8M urea was added and blown evenly, and the concentration was measured after centrifugation.

[0073] (2) Coating: The wall concentration and measurement value of the comparative antigen were compared, and the lower value was taken; the coating amount was 2 μg / ml, 10 ml / plate, and the coating antigen amount was prepared according to the actual use amount; the coated ELISA plate was labeled with antigen number, name, label, coating date and plate number, etc., and if the coating concentration was not 2 μg / ml, the coating concentration needed to be marked. After labeling the ELISA plate, 100 μl / well of coating antigen was added, and the plate was coated at 4°C overnight or at 37°C for 2 h.

[0074] (3) Washing plate: The coated plate was washed once with a plate washer and dried on absorbent paper. The 50X washing solution was prepared as follows: Tris 154.4 g, NaCl 149.0 g, Tween 20 24.0 ml, pure water 800 ml, about 45 ml of concentrated hydrochloric acid to adjust pH 7.2, and pure water to 1000 ml. Store at 4°C.

[0075] (4) Blocking: 2% skimmed milk powder was used as blocking solution for blocking, 200 μl / well, 4°C blocking overnight or 37°C blocking for 2 h.

[0076] (5) Washing plate: The blocked plate was washed once with a plate washer and dried on absorbent paper.

[0077] (6) Adding primary antibody: The corresponding primary antibody was added, the plasma was diluted with PBS at a ratio of 1:500, and incubated at 37°C for 1 h.

[0078] (7) Wash the plate with plate washer for more than 3 times, and dry on paper.

[0079] (8) Add secondary antibody (manufacturer: Biyun Tian; item number: A0201): since the primary antibody is derived from human plasma, the goat anti-human secondary antibody is added at 1:500, and incubated at 37°C for 1 hour.

[0080] (9) Wash the plate with plate washer for more than 3 times, and dry on paper. At the same time, prepare the color developing solution (TMB).

[0081] (10) Color development: after preparing the stop solution, add 100 μl / well of color developing solution. Shake the plate to accelerate the color development process and observe closely.

[0082] (11) TMB color development: when the color development reaches a certain degree (generally, the blank and negative controls should not be too high), add 50 μl / well of stop solution. After adding the stop solution, allow it to stand for more than 10 minutes to ensure complete termination and uniform color (shake the plate to accelerate the termination process). After termination, read the results

[0083] (12) Reading: the enzyme label must be preheated for more than 30 minutes; the TMB color development detection wavelength is 450 nm. Open the corresponding enzyme label measurement software and read the results. Store the data in the designated location and complete the data.

[0084] (13) Notes:

[0085] When adding samples to a 96-well plate using a gun, avoid generating bubbles and prevent the added sample from hanging on the wall.

[0086] After adding the sample, observe the entire plate to ensure that there are no bubbles at the bottom of the wells. If bubbles are found, shake the plate or use the gun tip to remove the bubbles.

[0087] Pay close attention when washing the plate to ensure complete and thorough washing.

[0088] Pay close attention when developing the color, and terminate the reaction in a timely manner based on the color development of the negative, blank, and positive controls.

[0089] (14) Formulas for various reagents

[0090] Coating solution: sodium carbonate-sodium bicarbonate buffer, pH 9.6: Na2CO3 1.59 g, NaHCO3 2.93 g, and purified water to 1000 ml. Finally, check the pH value with pH paper. Store at 4°C.

[0091] 2. ELISA result analysis

[0092] (1) Positive and negative judgment criteria: The average value (X) of negative serum samples, standard deviation (SD), and the upper limit of the confidence interval cut-off value are X+2SD. The OD value of the test sample at 450 nm is greater than or equal to X+2SD, which can be judged as positive, and less than X+2SD, which can be judged as negative.

[0093] (2) ROC curve: The MedCalc software (version 19.4.0) is used to draw the ROC curve (Receiver Operating Characteristic curve) to evaluate the optimal detection efficiency of the antigen.

[0094] (3) By calculation, 607 cases of B-ultrasound positive plasma (including suspected) and 636 cases of normal human plasma (determined to be negative) are detected using the hydatid antigen of the application, with a sensitivity of 96% and a specificity of 90%; while the same plasma is detected using commercial antigen, the sensitivity is 86% and the specificity is 92%. Figure 4 The ROC curve of PSAP-3 recombinant protein and commercial antigen Egr and the box plot of detection OD value are shown.

[0095] (4) Although the specificity of PSAP-3 recombinant protein is slightly lower than that of commercial antigen, the sensitivity of PSAP-3 recombinant protein is higher than that of commercial antigen, which shows that PSAP-3 recombinant antigen is likely to be a new hydatid antigen to replace commercial antigen for detecting hydatid disease. And the double feature ROC curve of PSAP-3 recombinant antigen and commercial antigen Egr is simulated by MedCalc software (logistic regression) (, Figure 5 ), the detection efficiency of the two antigens together is very good. Further shows that PSAP-3 recombinant antigen is likely to be a supplement to commercial antigen, and the samples that are not detected positive by commercial antigen are retested. SEQUENCE LISTING <110> Shenzhen Huada Kangle Gene Research Institute <120> A PSAP-3 protein fragment and its application in human hydatid disease <130> P22012952C <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Epitope 1 <400> 1 Pro Ser Asp Pro Asn Ser Arg Pro Pro Val Val 1 5 10 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Epitope 2 <400> 2 Lys Tyr Asp Cys Ser Lys Leu Ser Thr Arg Glu Glu Arg Cys Ser Asp 1 5 10 15 Glu Arg Leu Met Arg Leu Cys Gly 20 <210> 3 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Epitope 3 <400> 3 Pro Val Asp Pro Ser Asn Ala Cys Arg Phe Cys Arg Asn Leu Leu Thr 1 5 10 15 Gln Arg Leu Thr Tyr Gly Ser Phe Ala Leu Asp Cys Ser Ile Tyr 20 25 30 <210> 4 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Epitope 4 <400> 4 Lys Ile Arg Cys Gln Asn Val Arg Thr Leu Lys Val Pro Gly Arg Val 1 5 10 15 Ser Phe Asn Asn Leu Asp Ser Ile Cys Lys Glu Asn Gln Leu Cys Asp 20 25 30 Asp Ser Val Glu Ser Pro Pro Met Asn Ser Leu Ala Pro Leu Gln Lys 35 40 45 Ile Ile Gly Asp Asp Pro Cys Leu Trp Gly Pro Ser Val Ala Cys Arg 50 55 60 Asp Ala Asp Ile Ala Ala Arg Cys Gly Met Thr Thr Tyr Cys Gln His 65 70 75 80 His Val Trp Leu Ala Asp Gln Pro Gln Ser Arg Ala Glu Lys Val Pro 85 90 95 Ser Leu Ala Leu Ile Asp Cys Ser Arg Pro Leu Arg Glu Ile Cys Val 100 105 110 Ser Pro Leu Leu Arg Tyr Cys Gly Arg Ser Ile Leu Glu Gln Cys Arg 115 120 125 Leu Tyr Ala Ala Ser Gly Ser Val Gly Ser Asp Met Arg Arg Glu Met 130 135 140 Cys Lys Asp Arg Pro Leu Ser Phe Cys Ser Asp Pro Arg Met Val Lys 145 150 155 160 Leu Cys Gly Met Gly Glu Tyr Cys Glu Ala Met Ser Met Thr Pro Thr 165 170 175 Ser Pro Ala Pro Pro Asn Thr Glu Ala Pro Arg Asp Pro Arg Cys Arg 180 185 190 Leu Gly Ser Ala Phe Val Cys Gln Thr Tyr Ala Asn Ala Val Leu Cys 195 200 205 Asp Gln Val Glu Met Cys Arg Ser Arg Phe Trp Pro 210 215 220 <210> 5 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> PSAP-3 recombinant protein <400> 5 Pro Ser Asp Pro Asn Ser Arg Pro Pro Val Val Arg Lys Tyr Asp Cys 1 5 10 15 Ser Lys Leu Ser Thr Arg Glu Glu Arg Cys Ser Asp Glu Arg Leu Met 20 25 30 Arg Leu Cys Gly Phe Gly Ala Phe Cys Leu Gly Tyr Lys Arg Pro Pro 35 40 45 Val Asp Pro Ser Asn Ala Cys Arg Phe Cys Arg Asn Leu Leu Thr Gln 50 55 60 Arg Leu Thr Tyr Gly Ser Phe Ala Leu Asp Cys Ser Ile Tyr Val Asn 65 70 75 80 Pro Leu Glu Lys lie Arg Cys Gin Asn Val Arg Thr Leu Lys Val Pro 85 90 95 Gly Arg Val Ser Phe Asn Asn Leu Asp Ser lie Cys Lys Glu Asn Gin 100 105 110 Leu Cys Asp Asp Ser Val Glu Ser Pro Pro Met Asn Ser Leu Ala Pro 115 120 125 Leu Gin Lys lie lie Gly Asp Asp Pro Cys Leu Trp Gly Pro Ser Val 130 135 140 Ala Cys Arg Asp Ala Asp lie Ala Ala Arg Cys Gly Met Thr Thr Tyr 145 150 155 160 Cys Gin His His Val Trp Leu Ala Asp Gin Pro Gin Ser Arg Ala Glu 165 170 175 Lys Val Pro Ser Leu Ala Leu lie Asp Cys Ser Arg Pro Leu Arg Glu 180 185 190 lie Cys Val Ser Pro Leu Leu Arg Tyr Cys Gly Arg Ser lie Leu Glu 195 200 205 Gln Cys Arg Leu Tyr Ala Ala Ser Gly Ser Val Gly Ser Asp Met Arg 210 215 220 Arg Glu Met Cys Lys Asp Arg Pro Leu Ser Phe Cys Ser Asp Pro Arg 225 230 235 240 Met Val Lys Leu Cys Gly Met Gly Glu Tyr Cys Glu Ala Met Ser Met 245 250 255 Thr Pro Thr Ser Pro Ala Pro Pro Asn Thr Glu Ala Pro Arg Asp Pro 260 265 270 Arg Cys Arg Leu Gly Ser Ala Phe Val Cys Gln Thr Tyr Ala Asn Ala 275 280 285 Val Leu Cys Asp Gln Val Glu Met Cys Arg Ser Arg Phe Trp Pro Arg 290 295 300 Gly Val 305 <210> 6 <211> 921 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding PSAP-3 <400> 6 ccctcggatc caaactctcg tccgcccgtt gttcgcaagt acgactgcag taaactctcc 60 actcgtgagg aacgctgttc agacgagcgc ttgatgcgcc tgtgcggttt cggggccttt 120 tgcttggggt acaagcgtcc accggtggat ccctccaatg cctgccgttt ctgccgaaac 180 ctcctcactc aacgacttac ttacggtagc ttcgctcttg actgctccat ctatgtaaac 240 ccgttggaaa agatacgatg ccagaacgtt cgaacgctca aggttccggg gcgtgtgtct 300 tttaacaacc tcgattccat ttgcaaggaa aatcaactct gcgatgattc ggtagaatca 360 ccgccaatga actccctcgc tccccttcaa aaaatcatcg gtgatgatcc ctgtctctgg 420 ggtccctcgg tcgcgtgtcg cgatgctgat attgcagctc gttgtggtat gactacctac 480 tgccaacacc acgtctggct ggctgatcaa ccgcagtctc gagccgaaaa agttcccagc 540 ttggccctca ttgattgctc ccgtcccctg cgtgagatct gtgtctctcc attgttgcgc 600 tactgtggac gctctattct ggagcagtgt cgtctctacg ccgcgtcggg cagtgttggg 660 agtgatatgc gtcgtgaaat gtgcaaagac cgtcctctaa gcttctgttc agatcccagg 720 atggtaaaac tgtgtggaat gggcgagtac tgcgaggcaa tgagcatgac tcctacatca 780 ccagcaccgc cgaatacaga ggctccacgc gatcctcgat gtcgtctcgg ctcagctttt 840 gtatgtcaaa cctacgcaaa tgcagtgctc tgtgaccagg tggagatgtg ccgtagcaga 900 ttctggccac gaggagtgta g 921

Claims

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

5.

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

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

6.

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

5. A transformant characterized in that, It comprises the isolated nucleic acid as claimed in claim 2 or 3 or the recombinant expression vector as claimed in claim 4, wherein the transformant is a bacterium or a eukaryotic cell.

6. The transformant of claim 5, wherein, The bacteria are .

7. The transformant according to claim 5 or 6, wherein The amino acid sequence of the protein expressed by the transformant is shown as SEQ ID NO:

5.

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

9. The kit of claim 8, wherein The kit is an indirect ELISA detection kit.

10. The kit of claim 9, wherein The indirect ELISA detection kit further comprises a secondary antibody, a coating solution, a washing solution, a color developing solution, a termination solution and a dilution solution.

11. A marker combination, characterized in that The marker combination comprises the recombinant protein as claimed in claim 1.

12. The marker combination of claim 11, wherein, The marker combination further comprises one or more of Egr, antigen B and antigen 5.

13. A method of producing the recombinant protein according to claim 1, characterized by, It comprises the following steps: The transformant as claimed in any one of claims 5-7 is cultured under conditions suitable for its fermentation so that it expresses the recombinant protein.

14. Use of the recombinant protein as claimed in claim 1 or the marker combination as claimed in claim 11 or 12 in the preparation of an antibody against echinococcosis or in the preparation of a diagnostic agent for diagnosing diseases caused by hydatid cysts.

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