Novel echinococcus granulosus positive antigen and application thereof

Abstract

The invention provides a novel positive antigen of echinococcus granulosus and application of the positive antigen. Specifically, the invention provides an application of Echinococcus granulosus lactic dehydrogenase (EgLDH) or a gene thereof or a detection reagent thereof, and the EgLDH or the gene thereof or the detection reagent thereof is (i) used for preparing a positive antigen of the Echinococcus granulosus (or echinococcosis granulosus), and (ii) used for preparing a negative antigen of the Echinococcus granulosus (or echinococcosis granulosus), and (ii) used for preparing a negative antigen of the Echinococcus granulosus (or echinococcosis granulosus (or echinococcosis granulosus (or echinococcosis granulosus). And / or (ii) is used for preparing a reagent or a kit for diagnosing echinococcus granulosus (or echinococcus granulosus). The antigen disclosed by the invention has very high diagnostic sensitivity and specificity on echinococcus granulosus, and the diagnostic efficiency is far higher than that of other conventional echinococcus granulosus antigens.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of December 31, 2014, application number CN201410848967.5, and invention name “A new positive antigen of Echinococcus granulosus and its application”. Technical Field

[0002] The present invention relates to the field of parasitic disease diagnosis, and in particular to a novel antigen that can be used to diagnose Echinococcus granulosus (or Echinococcus granulosus). Background Art

[0003] Echinococcosis, also known as cystic hydatid disease, is caused by the larvae (echinococcosis) of the tapeworm Echinococcus granulosus. It is a zoonosis that seriously endangers human health and life safety, and impacts socioeconomic development. It is the leading cause of echinococcosis burden worldwide and a key parasitic disease targeted for prevention and control in my country. The disease has a global distribution, but reports indicate that Echinococcus granulosus is endemic in at least 100 countries worldwide. Highly endemic areas are primarily in Eurasia, northern and eastern Africa, Australia, and South America.

[0004] Currently, the diagnosis of echinococcosis is primarily based on patient-reported symptoms and a local physical examination by a physician. Because Echinococcus granulosus parasitizes internal organs in an enclosed manner, traditional etiological methods are ineffective and must be supplemented with physical and other experimental methods.

[0005] Current diagnosis relies primarily on medical imaging. Ultrasound is currently the primary method for diagnosing and investigating echinococcosis, but using ultrasound for disease screening still presents challenges. For example, it requires high technical expertise and experience from the examiner, can easily be confused with liver cancer, hemangiomas, and liver cysts, and can be difficult to accurately diagnose cysts smaller than 2 cm.

[0006] Immunological testing is an important auxiliary diagnostic method for human echinococcosis. When the imaging morphology of the cysts is atypical, immunological diagnosis has the significance of differential diagnosis; the sensitivity and specificity of immunological testing also play an important role in the timely detection and follow-up of patients after treatment; immunological diagnosis is of even greater reference value for epidemiological surveys in endemic areas, the formulation of prevention and control plans, and the assessment of prevention and control effects. The serological diagnosis of echinococcosis has a long history of development, and almost all existing immunological diagnostic methods have been used for the diagnosis of human echinococcosis. However, to date, there is no feasible diagnostic tool, kit, or method that can be widely applied in the clinical practice of echinococcosis. Improving the detection efficiency of serological diagnostic reagents for human echinococcosis is an urgent problem to be solved in the prevention and control of echinococcosis in my country.

[0007] Due to the low sensitivity of circulating antigen detection, antibody testing remains the most widely used method for clinical diagnosis and epidemiological surveys of echinococcosis. The sensitivity and specificity of antibody testing are closely related to the antigen used in the experiment. Antigens for echinococcosis can be derived from cyst fluid, protoscoleces, and cyst walls. Cyst fluid antigen (HCF) has been the most studied and is currently the primary source of diagnostic antigen. Cyst fluid has a complex composition, including parasite secretions and shed material from the parasite's surface, and possesses strong antigenicity. However, the antigenicity of cyst fluid varies significantly between different hosts and within different parasitic sites within the same host. While sensitivity has been reported to reach 75% to 95%, specificity is low and cross-reactivity with other tapeworms, nematodes, and flukes is common. Furthermore, the complexity of crude cyst fluid from natural sources makes quality control and standardization difficult, resulting in low production volumes. This is a key factor contributing to the low efficacy and lack of standardization of current serological diagnostic reagents for echinococcosis.

[0008] Analyzing crude antigen components and using genetic engineering methods to prepare recombinant antigens to replace natural antigens is an important approach to addressing these issues and improving the efficacy of diagnostic reagents for echinococcosis. Antigen B (AgB) is one of the most abundant components in the cyst fluid of Echinococcus granulosus. Recombinant proteins of its subunits, EgAgB1 and EgAgB2, are currently recognized as the most promising diagnostic antigens for echinococcosis. Currently reported natural antigen B has a certain sensitivity and specificity for the diagnosis of cystic echinococcosis, but the sensitivity is often low. While AgB-based peptide antigens, such as P176, P175, and P177, can improve diagnostic specificity, they often reduce sensitivity.

[0009] In addition, new diagnostic antigens have been discovered. However, the sensitivity and specificity of these reported antigens for detecting echinococcosis granulosus remain significantly limited in practical diagnostic applications, failing to meet diagnostic requirements. Therefore, there is an urgent need to develop antigens that can effectively improve the sensitivity and specificity of echinococcosis diagnosis. However, the use of Echinococcus granulosus LDH as a new, effective antigen for diagnosing echinococcosis granulosus has not yet been reported.

[0010] Therefore, there is an urgent need in the art to develop an antigen that can effectively improve the diagnostic sensitivity and specificity of Echinococcus granulosus. Summary of the Invention

[0011] The purpose of the present invention is to provide an antigen that can effectively improve the diagnostic sensitivity and specificity of echinococcosis, as well as its preparation method and application.

[0012] In a first aspect of the present invention, there is provided a use of Echinococcus granulosus lactate dehydrogenase (EgLDH) or its gene, or a detection reagent thereof, which is used (i) for preparing a positive antigen for Echinococcus granulosus (or Echinococcus granulosus larvae); and / or (ii) for preparing a reagent or kit for diagnosing Echinococcus granulosus (or Echinococcus granulosus larvae).

[0013] In another preferred embodiment, the Echinococcus granulosus lactate dehydrogenase EgLDH is selected from the following group:

[0014] (a) a polypeptide as shown in SEQ ID NO.: 1;

[0015] (b) an immunogenic derivative polypeptide formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 1;

[0016] (c) an immunogenic polypeptide having a homology of ≥90%, preferably ≥95%, to the amino acid sequence of SEQ ID NO: 1; or

[0017] (d) Fragments of the immunogenic polypeptides (a)-(c).

[0018] In another preferred embodiment, the antigens in the antigen combination are directed against different antibodies.

[0019] In another preferred embodiment, the GenBank accession number of the EgLDH gene is HM748917.1

[0020] In another preferred embodiment, the nucleotide sequence of the EgLDH gene is shown in SEQ ID NO.: 2.

[0021] In another preferred embodiment, the derivative polypeptide retains at least 80%, 90%, or 95% of the immunogenicity of the polypeptide shown in SEQ ID NO.: 1.

[0022] In another preferred embodiment, the derivative polypeptide is formed by substituting, deleting or adding 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10 amino acids of the polypeptide shown in SEQ ID NO.: 1, and the derivative polypeptide is immunogenic.

[0023] In another preferred embodiment, the detection reagent includes an antibody against Echinococcus granulosus lactate dehydrogenase, a probe for detecting the coding sequence of Echinococcus granulosus lactate dehydrogenase, or a combination thereof.

[0024] In another preferred embodiment, the kit comprises a container containing EgLDH or an antigen-antibody complex that specifically binds to EgLDH.

[0025] In another preferred embodiment, the container contains an antigen-antibody complex that specifically binds to EgLDH as a positive control.

[0026] In a second aspect of the present invention, an antibody is provided, wherein the antibody can specifically bind to the polypeptide shown in SEQ ID NO.: 1.

[0027] In a third aspect of the present invention, an antigen-antibody complex is provided, wherein the complex comprises:

[0028] (i) the polypeptide represented by SEQ ID NO.: 1;

[0029] (ii) an antibody that specifically binds to the polypeptide in (i).

[0030] In a fourth aspect of the present invention, there is provided a use of the antigen-antibody complex according to the third aspect of the present invention, which is used for:

[0031] (a) preparing a reagent or kit for detecting Echinococcus granulosus (or Echinococcus granulosus); and

[0032] (b) Used as a positive control for the detection of Echinococcus granulosus (or Echinococcus granulosus).

[0033] In a fifth aspect of the present invention, a kit for detecting Echinococcus granulosus (or Echinococcus granulosus) is provided, characterized in that the kit comprises a container containing Echinococcus granulosus lactate dehydrogenase or its gene.

[0034] In another preferred embodiment, the kit further comprises an enzyme binding solution, a reaction substrate and optional instructions. Preferably, the kit further comprises components selected from the following group: a reaction termination solution, a sample diluent, and a washing solution.

[0035] In a sixth aspect of the present invention, a method for diagnostically or non-diagnostally detecting whether a sample is infected with Echinococcus granulosus (or Echinococcus granulosus) is provided, comprising the steps of:

[0036] (1) preparing the Echinococcus granulosus lactate dehydrogenase represented by SEQ ID NO.: 1 as an antigen;

[0037] (2) contacting the antigen obtained in step (1) with the sample to be tested;

[0038] (3) detecting whether the sample contains the antigen-antibody complex described in the third aspect of the present invention;

[0039] Wherein, if the antigen-antibody complex appears in the sample in step (3), it indicates that the sample is infected with Echinococcus granulosus (or Echinococcus granulosus).

[0040] In another preferred embodiment, the samples include blood samples (including whole blood, serum, and plasma samples), tissue fluid samples, urine samples, saliva samples, and feces samples.

[0041] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The results of PCR amplification of the EgLDH gene are shown. The lanes in the figure are as follows: M: molecular weight standard, 1: EgLDH gene amplification product (indicated by the arrow).

[0043] Figure 2 The results of PCR amplification of E. coli transformed with the recombinant plasmid pET-28a-EgLDH are shown. Lanes in Panel A are as follows: M: molecular weight marker, 1, 3: bacteria carrying no plasmid, 2, 4: bacteria carrying the EgLDH gene (indicated by arrows); Lanes in Panel B are as follows: 1: pET28a-EgLDH plasmid amplification product (indicated by arrows), M: molecular weight marker.

[0044] Figure 3 The results of recombinant protein EgLDH expression and purification analysis are shown. The lanes in Figure A are as follows: M: standard molecular weight protein, 1: empty bacterial protein, 2: uninduced bacterial protein, 3: supernatant of induced bacterial lysis (the arrow indicates the target protein), 4: precipitate of induced bacterial lysis; the lanes in Figure B are as follows: M: standard molecular weight protein, 1: purified recombinant EgLDH (indicated by the arrow), 2: soluble bacterial protein containing EgLDH protein before purification

[0045] Figure 4 The ELISA results of EgLDH, HCF, and EgAgB1 antigens are shown.

[0046] Figure 5 A standard curve for protein concentration determination in one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0047] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that lactate dehydrogenase (EgLDH) from Echinococcus granulosus can serve as a highly immunogenic antigen for diagnosing Echinococcus granulosus, with very high sensitivity and specificity for the diagnosis of Echinococcus granulosus. Based on this invention, EgLDH can be used as a diagnostic antigen for Echinococcus granulosus and further used in the preparation of reagents or kits for diagnosing and detecting Echinococcus granulosus (or Echinococcus granulosus). This has led to the completion of the present invention.

[0048] LDH antigen

[0049] Lactate dehydrogenase (LDH) is an important functional enzyme widely present in animal, plant and microbial cells. It is one of the key enzymes in the glycolysis pathway. + With the assistance of LDH, LDH can catalyze the reversible reaction between pyruvate and lactate, releasing energy for the body's needs.

[0050] In the present invention, the Echinococcus granulosus LDH is used as an antigen for the first time as a diagnostic marker for echinococcosis, and experiments have shown that the Echinococcus granulosus LDH helps to significantly improve the sensitivity and specificity of echinococcosis diagnosis.

[0051] The amino acid sequence and nucleotide sequence information of Echinococcus granulosus LDH are as follows:

[0052] Amino acid sequence of EgLDH:

[0053] MSVEGLLLPLEMEQCFGRERKVSVVGAGAVGTAAVFAIMTKGIANTVALYDIDEDRCNGEVMDLDQGSLFLESCRVIGGKDITKTADSDIVVVTAGARQAVGESRLNLVQRNVDIFKKLIPTLVEQSPKCILVIVTNPVDIMTYVSWKLSGFPQHRVLGSGTMLDTA RFRHILGEKLNVHPSAIHGYVVGEHGDSSVPVWSRVTVGGANLCDIYPKIGQAGDPDDFASIHKAVVDSAYEIIRMKGCTAWAIGLCCASLCNAILRNKKIVIPVSTSLKGKLGIKEEVFTSVPCIVDSSGVSAVINLEYSPSEKQSLLASVETLQKIIAGIKW(SEQ ID NO.:1)

[0054] EgLDH nucleotide sequence:

[0055] Atgtctgtggaggggttgttgttgcctttggagatggaacagtgttttgggcgtgagcggaaggtttctgttgttggtgcgggagcagtaggcacggcagcggtgtttgctattatgactaaaggtattgcaaacactgtcgctctctacgatattgacgaagatagatgcaacggtgaagtgatggacttggaccaaggctcactgtttctggagtcttgtagagtaattggtggcaaagatataacgaagactgcggactcggatatcgttgtagtaacagctggggcccggcaagctgttggcgaatccagattgaaccttgttcaacgcaatgttgatatatttaaaaaactaattcctactctcgttgaacaaagcccaaagtgcattctcgttatcgttacaaatccagttgatatcatgacctatgtctcctggaagttaagcggctttccacagcatcgtgtcttaggatctggaaccatgcttgacactgctagatttcggcacattcttggcgagaagttgaatgtgcatcctagtgccatacacggttacgtagtcggcgaacatggagactcgagtgtgccagtatggagtagggtgaccgttggtggagcaaatctctgtgacatttatcccaagatcggccaagccggcgatcccgacgattttgcttccattcacaaggctgttgtcgatagcgcctacgaaatcattcgcatgaagggctgcactgcttgggccataggtctctgttgcgcctccctgtgtaatgcgattctgcgaaacaagaaaattgtgattccagtttccacctcacttaagggcaaacttggtatcaaagaggaggtgttcacgagcgtgccctgtatagtcgacagcagcggcgtgtctgcagtgatcaacctcgagtattcgcctagtgagaaacaaagcctgttggccagtgttgagactcttcagaagattatcgcaggcatcaagtggtga(SEQID NO.:2)

[0056] Positive antigen

[0057] In this application, the terms "antigen" and "positive antigen" are used interchangeably to refer to proteins or polypeptides that specifically bind to antibodies against Echinococcus granulosus. An antigen is a substance that can stimulate a (specific) immune response and bind to immune response products, antibodies, and sensitized lymphocytes in vitro to produce an immune effect (specific reaction). Antigens have two basic properties: the ability to induce an immune response, or immunogenicity, and the ability to react with immune response products, or antigenicity.

[0058] As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and proteins in their natural state within living cells are not isolated and purified, but the same polynucleotides or proteins are isolated and purified if they are separated from other substances that exist with them in their natural state. "Isolated positive antigen" means that the antigen is essentially free of other proteins, lipids, carbohydrates or other substances that are naturally associated with it. Those skilled in the art can purify positive antigens using standard protein purification techniques. Substantially pure protein antigens can produce a single main band on a non-reducing polyacrylamide gel. The purity of the antigen can be analyzed by amino acid sequence.

[0059] Antigens of the present invention can be naturally purified products, or chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, antigens of the present invention can be glycosylated or non-glycosylated. Antigens of the present invention may or may not include an initial methionine residue.

[0060] The present invention also includes fragments, derivatives and analogs of antigens. As used herein, the terms "fragment," "derivative," and "analog" refer to antigens that substantially retain the same biological function or activity as the native antigens of the present invention. The antigen fragments, derivatives, or analogs of the present invention may be (i) antigens in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) antigens having a substituent group in one or more amino acid residues, or (iii) antigens formed by fusion with another compound (such as a compound that extends the half-life of the antigen, such as polyethylene glycol), or (iv) antigens formed by fusion of an additional amino acid sequence to the antigen sequence. These fragments, derivatives, and analogs are well known to those skilled in the art.

[0061] In the present invention, the term "antigen" also includes variant forms with the same function. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions, and / or substitutions, as well as the addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein.

[0062] Modifications (usually without altering the primary structure) include: chemical derivatization of the antigen in vivo or in vitro such as acetylation, carboxylation, glycosylation, and sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

[0063] Antigen coding sequence

[0064] The polynucleotide encoding the antigen of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The term "polynucleotide encoding the antigen of the present invention" may include a polynucleotide encoding the antigen or a polynucleotide further comprising additional coding and / or non-coding sequences.

[0065] The present invention also relates to variants of the polynucleotides described above. Variants of the polynucleotides described above can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. The present invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions.

[0066] The nucleotide sequences of the present invention or their fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is typically accomplished by cloning them into vectors, transferring them into cells, and then isolating the relevant sequences from the propagated host cells by conventional methods. In addition, artificial synthesis methods can also be used to synthesize the relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. DNA sequences encoding the antigens of the present invention (or their fragments, or their derivatives) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the antigen sequences of the present invention by chemical synthesis.

[0067] The method of amplifying DNA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods.

[0068] Antigen preparation method

[0069] The method for preparing the antigen of the present invention comprises the following steps:

[0070] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the positive antigen of the present invention, or a recombinant expression vector containing the polynucleotide;

[0071] (2) Host cells cultured in a suitable culture medium;

[0072] (3) Isolate and purify the antigen of the present invention from the culture medium or cells.

[0073] In the present invention, the polynucleotide sequence can be inserted into a recombinant expression vector. Methods well known to those skilled in the art can be used to construct an expression vector containing an antigen-encoding DNA sequence and appropriate transcription / translation control signals. Vectors containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable protein expression.

[0074] Host cells can be prokaryotes, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2 using procedures well known in the art. Another method uses MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

[0075] The transformants obtained can be cultured using conventional methods to express the antigenic protein of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, the promoter selected is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0076] Antigen protein can be expressed in the cell or on the cell membrane or secreted outside the cell. If necessary, the antigen protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and the combination of these methods.

[0077] Reagent test kit

[0078] The present invention also provides a kit for detecting Echinococcus granulosus. Generally, the kit of the present invention includes the following components: a container or a carrier; and the EgLDH protein, gene or its detection reagent located in the container or on the carrier. In another preferred embodiment, the kit also includes an enzyme conjugate solution, a reaction substrate and optional instructions. Preferably, the kit may also include components selected from the following group: a reaction stop solution, a sample diluent, and a washing solution. A preferred kit for detecting Echinococcus granulosus includes: a container and a coating solution located in the container, a horseradish peroxidase-labeled mouse (or sheep) anti-human IgG (H+L) antibody, a substrate solution TMB, a dilute concentrated sulfuric acid reaction stop solution and a sample diluent. More preferably, it also contains a blank control, positive and negative controls and a control to monitor whether the detection process meets the standards.

[0079] The main advantages of the present invention are:

[0080] 1. Antigen has high sensitivity and is not affected by the infection of the test sample, so it has high sensitivity.

[0081] 2. High specificity: the antigen of the present invention detects the sample to be tested with an extremely low false positive rate.

[0082] 3. Easy to operate, the fluorescence intensity value of the antigen can be read using the universal ELISA method.

[0083] 4. The results are stable and highly repeatable.

[0084] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are calculated by weight.

[0085] General methods and materials

[0086] Main reagents:

[0087]

[0088]

[0089] Main instruments:

[0090]

[0091] Test materials:

[0092] Echinococcus granulosus cDNA, Escherichia coli DH5a, Escherichia coli BL21 (DE3), plasmid pET-28a, serum from patients with Echinococcus granulosus and healthy controls for evaluation, cyst fluid antigen (HCF), and EgAgB1 polypeptide antigen were all provided by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention.

[0093] Example 1: Cloning of the lactate dehydrogenase (EgLDH) gene from Echinococcus granulosus

[0094] 1.1 Amplification of EgLDH gene:

[0095] 1.1.1 Target gene primer design:

[0096] Primers were designed using PrimerPremier 5 software based on the known full-length sequence encoding EgLDH (GenBank accession number: HM748917.1):

[0097] EgLDH-F: The italicized sequences are homologous to the vector, and the underlined ones are XhoI restriction enzyme cutting sites.

[0098] EgLDH-R: The italics indicate sequences homologous to the vector, and the underline indicates the BamHI restriction site (synthesized by BGI).

[0099] 1.1.2 PCR amplification of target gene:

[0100] Using Echinococcus granulosus cDNA as a template, PCR amplification of the target gene was performed. The PCR reaction system was as follows:

[0101]

[0102] The reaction conditions are as follows:

[0103]

[0104] The PCR products were electrophoresed on a 1.2% agarose gel at 130V for approximately 30 minutes. Figure 1 The PCR results showed a clear target band at approximately 966 bp, which was consistent with the expected fragment size, indicating that the EgLDH target gene was successfully amplified.

[0105] 1.1.3 Target gene purification:

[0106] Using QIAGEN The gel recovery kit is used to purify the target gene amplified by PCR. The specific operation is as follows:

[0107] (1) After electrophoresis, use a clean, sharp blade to cut the target DNA fragment from the agarose gel.

[0108] (2) Crush the cut gel pieces and place them in a centrifuge tube and weigh them. Add Buffer QG 3 times the volume of the gel.

[0109] (3) Place the centrifuge tube in a 50°C water bath and incubate for 10 minutes. To accelerate the dissolution of the gel, take out the centrifuge tube and invert it every 2-3 minutes to mix it thoroughly.

[0110] (4) After the gel is completely dissolved, observe whether the color of the liquid in the centrifuge tube is basically the same as the color of Buffer QG before dissolution. If the liquid in the tube turns orange or purple, add 10 μl of 3M sodium acetate (pH 5.0) to adjust the pH value.

[0111] (5) Add 1 times the volume of gel to the centrifuge tube and mix well.

[0112] (6) Place the QIAquick spin column in the kit into a 2 ml collection tube; add the liquid in the centrifuge tube to the QIAquick spin column, centrifuge at 13,000 rpm for 1 min, and discard the filtrate.

[0113] (7) Return the QIAquick spin column to the original centrifuge tube, add 500 μl of Buffer QG, centrifuge at 13,000 rpm for 1 min, and discard the filtrate.

[0114] (8) Add 750 μl of Buffer PE to the QIAquick spin column to wash the column. Let it stand for 2-5 minutes, centrifuge at 13,000 rpm for 1 minute, and discard the filtrate.

[0115] (9) Centrifuge again at 13000 rpm for 2 min to remove residual Buffer PE.

[0116] (10) Place the QIAquick spin column in a new 1.5 ml centrifuge tube; add 30 μl of Buffer EB to the center of the QIAquick spin column membrane, let it stand for 4 minutes, centrifuge at 13,000 rpm for 1 minute, collect the eluate, and store at -20°C.

[0117] (11) The recovery efficiency was detected by agarose gel electrophoresis and the concentration of DNA fragments was estimated.

[0118] Example 2: Linearization of vector

[0119] 2.1 Preparation of empty plasmid pET-28a:

[0120] The pET-28a plasmid carrier bacteria (Novagen) were plated, and after PCR identification of single colonies, the plasmid was extracted and linearized by double enzyme digestion as follows:

[0121] (1) Dilute 1 μl of the seed-preserving vector bacterial solution with 500 μl of ddH2O, and spread 100 μl onto Kana-resistant LB solid medium. Incubate at 37°C for approximately 1 hour until the surface liquid is absorbed. Invert the plate and incubate at 37°C for 16 hours.

[0122] (2) Randomly pick 4 single colonies and pipette them into 20 μl of LB culture medium. Take 3 μl as template and perform PCR verification. The system is as follows:

[0123]

[0124] Reaction conditions:

[0125]

[0126] After the reaction was completed, the PCR products were subjected to 1% agarose gel electrophoresis (2 μl EB substitute / 40 ml) at 130 V for 25 min.

[0127] (3) Add the bacterial solution identified correctly by PCR to 5 ml of Kana-resistant LB culture medium, mix well, and place in a shaker at 37°C, 200 rpm / min, and culture for 12-16 hours.

[0128] (4) Extract the pET-28a vector according to the instructions of the Axygen plasmid DNA miniprep kit as follows:

[0129] ① Take about 4 ml of overnight culture solution, centrifuge at 12,000 × g for 1 min, and discard the supernatant;

[0130] ② Add 250 μl Buffer S1 to suspend the bacterial pellet and pipette evenly to ensure no small bacterial clumps remain;

[0131] ③ Add 250 μl of Buffer S2 and gently invert the tube 4-6 times to mix evenly to fully lyse the bacteria. This process should not exceed 5 minutes.

[0132] ④ Add 350 μl of Buffer S3, gently invert up and down 6-8 times to mix, and centrifuge at 12,000 × g for 10 min;

[0133] ⑤ Transfer the supernatant from centrifugation step ④ to a preparation tube (placed inside a centrifuge tube), centrifuge at 12,000 × g for 1 min, and discard the filtrate;

[0134] ⑥ Place the prepared tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate;

[0135] ⑦ Place the prepared tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. Repeat this step once.

[0136] ⑧Place the above preparation tube back into the centrifuge tube and centrifuge at 12,000×g for 1 min without rotation;

[0137] ⑨ Place the preparation tube in a new 1.5ml centrifuge tube, add 30μl Eluent to the center of the preparation tube, let it stand at room temperature for 1 minute, and centrifuge at 12,000×g for 1 minute.

[0138] ⑩ Determine the plasmid concentration.

[0139] 2.2 Double enzyme digestion of empty plasmid pET-28a:

[0140] (1) The plasmid extracted in step (4) was double-digested with Xho I / Bam HI:

[0141]

[0142] (2) Purify the plasmid product after double enzyme digestion according to the instructions of the EZNA Ultra-Sep Gel Extraction Kit as follows:

[0143] ① Separate DNA fragments by agarose gel electrophoresis.

[0144] ② After the fragments are completely separated, quickly cut the required bands under ultraviolet light. The DNA exposure time under ultraviolet light should not exceed 30 seconds.

[0145] ③ Weigh the gel block and add 1 ml of Binding Buffer per 1 g of gel. Add an appropriate volume of Binding Buffer and incubate in a 55-60°C water bath until the gel is completely dissolved (approximately 7-10 minutes). Oscillate every 2-3 minutes.

[0146] ④Place the HiBind DNA column in a 2ml collection tube.

[0147] ⑤ Transfer the DNA / gel mixture to a HiBind DNA column in a 2 ml collection tube and centrifuge at 10,000 × g for 1 min.

[0148] ⑥ Pour off the filtrate and put the column back into the collection tube.

[0149] ⑦Reinstall the column into the collection tube, add 300ul Binding Buffer, centrifuge according to the above conditions, and discard the filtrate.

[0150] ⑧Replace the column into the collection tube, add 700 μl of SPW Wash Buffer, centrifuge as described above, and discard the filtrate. Note: Wash Buffer must be diluted with anhydrous ethanol before use. Repeat this step once.

[0151] ⑨ Discard the filtrate, reinstall the column into the collection tube, and centrifuge the empty column at 13,000×g for 2 minutes to dry the column matrix.

[0152] ⑩ Place the column in a clean 1.5ml centrifuge tube and add 30-50μl of Elution Buffer preheated at 65°C to the column matrix. Let it stand at room temperature for 2 minutes. Centrifuge at 13,000×g for 2 minutes to elute the DNA.

[0153] The linearized plasmid digested with enzymes was purified and recovered according to the method in 1.13 and used for the construction of the recombinant plasmid in 3.1.

[0154] Example 3: Construction and identification of recombinant plasmids

[0155] 3.1 Construction and transformation of recombinant plasmid:

[0156] Use In- The HD Cloning Kit seamlessly clones the gene successfully amplified in 1.1 and the plasmid linearized in 2.2. The steps are as follows:

[0157] (1) Add 2 μl of Cloning Enhancer to 5 μl of PCR product.

[0158] (2) Place the above mixture in a PCR instrument and react at 37°C for 15 minutes, then at 80°C for 15 minutes.

[0159] (3) The cloning reaction system is as follows:

[0160]

[0161] (4) Place the prepared reaction system in a PCR instrument, incubate at 37°C for 15 min, then at 50°C for 15 min, then remove and place on ice.

[0162] (5) Add the above system to a centrifuge tube containing 100 μl of DH5α competent cells, pipette slowly 3 to 5 times, and place on ice for 30 minutes.

[0163] (6) Place the centrifuge tube containing the DH5a competent cells in a 42°C water bath for 60 seconds, then quickly place it in an ice bath for 2 minutes.

[0164] (7) Add 900 μl of LB medium without resistance to each tube of DH5a competent cells, mix well, and incubate at 37°C with shaking at 150 rpm / min for 45 min.

[0165] (8) Centrifuge at 5000×g for 30 seconds, take 800 μl of the supernatant, pipette and mix the precipitate, and plate onto kanamycin-resistant LB solid medium. Incubate upright at 37°C for 1 hour until the liquid is absorbed. Invert the plate and incubate at 37°C overnight.

[0166] (9) Randomly pick 4 single colonies from each plate and add 3 μl of 20 μl LB culture medium as a template for colony PCR verification. The reaction system is as follows:

[0167]

[0168] The reaction conditions are as follows:

[0169]

[0170] The PCR reaction products were electrophoresed on a 1.2% agarose gel at 130V for about 30 minutes. The results showed that the target gene was successfully cloned. Figure 2 shown.

[0171] (10) Add the bacterial suspension with the correct band size to 3 ml of LB culture medium containing kanamycin and culture overnight at 37°C with shaking at 200 rpm / min.

[0172] (11) Take 700 μl of bacterial solution and add 300 μl of 50% glycerol and store at -80°C.

[0173] 3.2 Extraction and identification of recombinant plasmid:

[0174] The recombinant bacteria identified correctly by colony PCR were subjected to plasmid DNA extraction using the AxyPrep Plasmid DNA Mini Kit (Axygen, USA). The specific operation process is as follows.

[0175] (1) Take approximately 4 ml of overnight culture and centrifuge at 12,000 × g for 1 min. Discard the supernatant. Optimization 1: Prepare two parallel tubes of culture for each recombinant plasmid.

[0176] (2) Add 250 μl of Buffer S1 to suspend the bacterial pellet and pipette evenly to ensure that no small bacterial clumps remain.

[0177] (3) Add 250 μl of Buffer S2 and gently invert the tube 4-6 times to mix evenly to fully lyse the bacteria.

[0178] (4) Add 350 μl of Buffer S3, gently invert up and down 6-8 times to mix, and centrifuge at 12,000 × g for 10 min.

[0179] (5) Transfer the supernatant from (4) to a preparation tube (placed inside a centrifuge tube), centrifuge at 12,000 × g for 1 min, and discard the filtrate. Optimization 2: Transfer the supernatant from the two tubes of bacterial lysis corresponding to each recombinant plasmid to the same preparation tube.

[0180] (6) Place the prepared tube back into a centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate.

[0181] (7) Place the prepared tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. Repeat this step once.

[0182] (8) Place the prepared tube back into the centrifuge tube and centrifuge at 12,000 × g for 1 min.

[0183] (9) Place the preparation tube in a new 1.5 ml centrifuge tube, add 30 μl of Eluent to the center of the preparation tube, let it stand at room temperature for 1 min, and centrifuge at 12,000 × g for 1 min.

[0184] (10) The plasmid concentration was determined and sent to BGI for sequencing to check whether the inserted fragment sequence was correct. The sequencing analysis results showed that the inserted fragment sequence was correct and the recombinant plasmid pET-28a-EgLDH was successfully constructed.

[0185] Example 4: Expression and identification of recombinant plasmids in E. coli

[0186] 4.1 Expression and identification of recombinant plasmid pET-28a-EgLDH in E. coli

[0187] 4.1.1 Transformation of recombinant plasmid and expression of target protein:

[0188] (1) About 40 ng of the correctly sequenced recombinant plasmid pET-28a-EgLDH was transformed into Escherichia coli BL21 (DE3) competent cells and cultured at 37°C overnight (the transformation method was the same as in Example 3.1).

[0189] (2) The next day, three single colonies were randomly picked and inoculated into 3 ml LB (containing 50 μg / L kanamycin) and cultured at 37°C and 180 rpm until the OD600 was approximately 0.8-1.0.

[0190] (3) Take out 300ul and add 700ul of glycerol as the bacterial culture, and take 1ml as the pre-induction control. Add IPTG with a final concentration of 1mM to the remaining bacterial liquid for induction. Continue to culture at 37℃, 200rpm for 4h.

[0191] (4) Take 10 μl of each bacterial suspension before and after induction, add 10 μl of 2X SDS-PAGE loading buffer, mix well, and boil at 100°C for 5 min to denature. Add 15 μl of sample to each sample and perform SDS-PAGE analysis. The separating gel is 12% and the stacking gel is 5%. The gel formula is as follows:

[0192] 12% separation gel

[0193]

[0194]

[0195] 5% separating gel

[0196]

[0197] The voltage settings were: 80V for 30 minutes for the separating gel and 120V for 60 minutes for the stacking gel. After removing the gel, stain it with staining solution for 1 hour, then destain it with destaining solution until the protein bands were clearly visible.

[0198] (5) Figure 3 As shown in Figure A, after IPTG induction, a clear expression band with a molecular weight of approximately 36 kDa appeared in the bacteria before induction, which is consistent with the expected protein size. This is the protein expressed by the recombinant plasmid pET-28a-EgLDH. The recombinant protein EgLDH expression strain was successfully constructed.

[0199] 4.1.2 Identification of recombinant protein solubility:

[0200] (1) Pipette 1.5 μl of the recombinant bacterial suspension constructed in 4.1.1 into 15 ml LB medium (containing 50 μg / L kanamycin) and culture at 37°C and 165 rpm for 7 h.

[0201] (2) Transfer the cells to 500 ml of LB medium (plus 50 mg / L kanamycin) and continue culturing at 37°C and 165 rpm until the OD600 is approximately 1.

[0202] (3) Add IPTG to a final concentration of 1 mM for induction and continue culturing at 37°C, 200 rpm for 4 h.

[0203] (4) Pipette 3 ml of the cultured bacteria and centrifuge at 12,000 rpm for 2 min to collect the bacteria. Centrifuge the remaining bacteria at 5,000 rpm for 20 min at 4°C. Discard the supernatant and resuspend the pellet in 20 ml of 1xPBS. Centrifuge again (same as above) to collect the pellet and freeze at -25°C for later use.

[0204] (5) Add 50 μl of BugBuster protein extraction reagent (Novagen) and nuclease (1‰) to the bacterial pellet collected from 3 ml of bacterial solution, fully resuspend the pellet, and lyse it on a shaker at room temperature for 1 h.

[0205] (6) Centrifuge 4 μl of bacterial lysate at 12,000 rpm for 10 min to separate the supernatant and precipitate. Resuspend the precipitate with an appropriate volume of 1xPBS, take out 5 μl of the supernatant and resuspended precipitate, mix them with 2xSDS-PAGE loading buffer, and then perform SDS-PAGE gel to verify the solubility of the expressed protein. The results showed that the recombinant protein was soluble. Figure 3 As shown in A (the arrow indicates the target protein).

[0206] Example 5: Purification of recombinant protein

[0207] 5.1 Purification of recombinant protein:

[0208] (1) Take the bacterial pellet collected in step 4.1.2 (4), add protease inhibitors, fully resuspend the pellet with Tris-HCl buffer (pH 7.4), and then sonicate: sonicate for 3 seconds, with an interval of 6 seconds, for a total of 10 minutes.

[0209] (2) Centrifuge the lysate at 4°C, 13,000 rpm for 45 min, collect the supernatant and filter through a 0.22 μmol filter.

[0210] (3) Ni-NTA affinity chromatography column was used for purification, and impurity proteins were removed with 50 mmol / L and the target protein was eluted with 500 mmol / L imidazole, and the elution fluid was collected.

[0211] (4) The collected purified protein was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). Figure 3 As shown in B (the arrow indicates the target protein), the results show that the purification effect is more than 90%.

[0212] 5.2 Determination of purified protein concentration:

[0213] The protein concentration was determined using the BCA protein quantification kit (Tiangen) according to the instructions:

[0214] (1) Dilution of standards: Dilute the BSA standard sample with 1x PBS to 1 mg / ml, 0.75 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.025 mg / ml and blank as a control.

[0215] (2) Prepare BCA working solution: Based on the number of samples, mix reagent A and reagent B in a volume ratio of 50:1 to prepare an appropriate volume of working solution and mix thoroughly.

[0216] (3) Take 20 μl of the diluted standard sample and the sample to be tested, add them to a 96-well plate, add 200 μl of working solution to each well, mix thoroughly, and incubate at 37°C for 30 min.

[0217] (4) Detect its absorbance at 562 nm using an ultraviolet spectrophotometer.

[0218] (5) Draw a standard curve using EXCEL based on the standard protein concentration and the corresponding absorbance value, and obtain the calculation formula for the protein concentration of the sample to be tested based on the standard curve, such as Figure 5 shown.

[0219] (6) According to Figure 5 The protein concentration of the sample (ie, purified EgLDH) was calculated to be 0.166 / ml using the calculation formula shown.

[0220] Example 6 Antigenicity Identification of Recombinant Purified Protein:

[0221] 6.1 ELISA evaluation of recombinant EgLDH antigen:

[0222] (1) Use coating solution to dilute recombinant EgLDH antigen to 39.5 μg / ml, cyst fluid antigen (HCF) to 10 μg / ml, and AgB1 polypeptide antigen to 2.19 μg / ml. The diluted antigens are coated on 96-well ELISA plates at 100 μl / well at 4°C overnight.

[0223] (2) The next day, the plate was washed three times with PBST, each time for 5 minutes, and then 2% BSA (100 μl / well) was added and blocked at 37°C for 2 hours.

[0224] (3) Wash the plate three times with PBST for 5 minutes each time, then add 1:100 diluted patient serum and normal human serum, 100 μl / well, and incubate at 37°C for 1 hour. Each plate should also have a positive control, a negative control, and a blank control.

[0225] (4) Wash the plate three times with PBST for 5 minutes each time, then add 100 μl / well of 1:10000 diluted HRP-labeled goat anti-human IgG antibody (Sigma) and incubate at 37°C for 1 hour.

[0226] (5) Wash the plate three times with PBST for 5 minutes each time, then add 100 μl / well of TMB substrate solution (protected from light). After color development at room temperature, add 2M concentrated sulfuric acid (100 μl / well) to terminate the reaction.

[0227] (6) Use an enzyme-labeled instrument to read the value at 450 nm. The result is as follows Figure 4 .

[0228] (7) Negative serum OD 450 The mean plus 2 times the standard deviation (M+2×SD) is the cut-off value, and the sensitivity, specificity, and Youden index (i.e., accuracy index) of the recombinant antigen protein are calculated. Among them, sensitivity = (number of true positive sera / number of patient sera) × 100%; specificity (specificity = number of true negative sera / number of normal human sera) × 100%; Youden index = sensitivity + specificity - 1

[0229] (8) The test results are listed in Table 1.

[0230] Table 1 ELISA test results of recombinant EgLDH, EgAgB1 and cyst fluid antigen (HCF)

[0231]

[0232] The cut-off value of ELISA detection for recombinant EgLDH was 0.195, with a sensitivity of 88%, a specificity of 98%, and an equivalent index of approximately 0.86; the cut-off value of ELISA detection for cyst fluid antigen (HCF) was 0.226, with a sensitivity of 93%, a specificity of 94%, and an equivalent index of approximately 0.87; the cut-off value of ELISA detection for EgAgB1 polypeptide antigen was 0.106, with a sensitivity of 68%, a specificity of 96%, and an equivalent index of approximately 0.64.

[0233] Comparative Example 1

[0234] Calreticulin (CRT) is one of the major calcium-binding proteins in the endoplasmic reticulum. It is present in all cells except red blood cells and has a strong calcium-binding capacity. The highly conserved genetic composition and amino acid sequence of calreticulin suggest its crucial role in maintaining cellular function. Recent studies have revealed a close relationship between calreticulin and immunity to parasitic infections, including its anticoagulant effects, participation in host immune responses, involvement in host autoimmunity, and immune evasion.

[0235] In addition to conducting ELISA evaluation on EgLDH, HCF, and EgAgB1 mentioned in the present invention, the inventors also used the cloning and expression methods mentioned in the present invention to prepare recombinant proteins for dozens of proteins, including the calreticulin gene of Echinococcus granulosus (GenBank accession number: EF587757.1), and obtained recombinant proteins with an electrophoretic purity of ≥90%.

[0236] Analysis of these various recombinant Echinococcus granulosus proteins using the same serum samples used in the present invention revealed that the vast majority of candidate proteins lacked sufficient sensitivity and specificity. For example, ELISA evaluation of the calreticulin antigen revealed that this recombinant antigen had a sensitivity of only 56%, a specificity of 96%, and a Youden Index of 0.52 for detecting echinococcosis granulosus. In contrast, the Youden Index of EgLDH, under the same experimental conditions, was as high as 0.86. The diagnostic efficacy of the recombinant EgLDH antigen in the present invention far surpasses that of recombinant Echinococcus granulosus calreticulin as an antigen for detecting echinococcosis granulosus.

[0237] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0238]

[0239]

Claims

1. A non-diagnostic method for detecting whether a sample is infected with Echinococcus granulosus, characterized in that: Including steps: (1) preparing Echinococcus granulosus lactate dehydrogenase (EgLDH) represented by SEQ ID NO.: 1 as an antigen; (2) contacting the antigen obtained in step (1) with the sample to be tested; (3) Detecting whether the sample contains antigen-antibody complexes; Wherein, if the antigen-antibody complex appears in the sample in step (3), it indicates that the sample is infected with Echinococcus granulosus. Wherein, the sample is a blood sample.

2. The method according to claim 1, wherein The blood sample includes a whole blood sample, a serum sample or a plasma sample.

3. The method according to claim 1, wherein The EgLDH is a recombinant protein EgLDH, which is produced from a prokaryotic host using recombinant technology, and the prokaryotic host is Escherichia coli.

4. The method according to claim 3, wherein The recombinant protein EgLDH is a soluble expressed protein.

5. The method according to claim 1, wherein The method further comprises: contacting the recombinant polypeptide EgAgB1 or EgAgB2 as an antigen with the sample to be tested, and detecting whether the sample contains an antigen-antibody complex; Alternatively, the method further comprises: contacting the sample to be tested with cyst fluid antigen (HCF) as an antigen, and detecting whether the sample contains an antigen-antibody complex.

6. The method according to claim 1, wherein The method further comprises: detecting and evaluating by ELISA; and using negative serum OD 450 The mean plus 2 times the standard deviation (M+2×SD) was the cut-off value, and the sensitivity, specificity and approximate index of the recombinant antigen protein were calculated; Among them, sensitivity = (number of true positive sera / number of patient sera) × 100%; specificity (specificity = number of true negative sera / number of normal human sera) × 100%; Youden index = sensitivity + specificity - 1.

7. A kit for detecting Echinococcus granulosus (or Echinococcus granulosus), characterized in that: The kit comprises a container, wherein the container contains: 1) A recombinant protein of Echinococcus granulosus lactate dehydrogenase, wherein the amino acid sequence of the recombinant protein EgLDH is shown in SEQ ID NO.: 1, and the recombinant protein EgLDH is produced using recombinant technology from a prokaryotic host, wherein the prokaryotic host is Escherichia coli, and the recombinant protein EgLDH is a soluble expressed protein; The container also contains an antigen-antibody complex that specifically binds to EgLDH as a positive control.

8. The detection kit according to claim 7, wherein The amino acid sequence of EgLDH is shown in SEQ ID NO.:

1.

9. The detection kit according to claim 7, wherein The kit also includes an enzyme binding solution, a reaction substrate and optional instructions; In addition, the kit further comprises the following components: a reaction termination solution, a sample diluent, and a washing solution.

10. A use of a recombinant Echinococcus granulosus lactate dehydrogenase (EgLDH), characterized in that: (i) for preparing a positive antigen for Echinococcus granulosus; and / or (ii) for preparing a reagent or kit for detecting Echinococcus granulosus, The amino acid sequence of the EgLDH is shown in SEQ ID NO.: 1, and the recombinant protein EgLDH is produced from a prokaryotic host using recombinant technology, the prokaryotic host is Escherichia coli, and the recombinant protein EgLDH is a soluble expressed protein.

Images