WO2015172706A1 - Criblage d'antigènes de diagnostic de schistosoma mansoni et utilisation associée - Google Patents

Criblage d'antigènes de diagnostic de schistosoma mansoni et utilisation associée Download PDF

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WO2015172706A1
WO2015172706A1 PCT/CN2015/078766 CN2015078766W WO2015172706A1 WO 2015172706 A1 WO2015172706 A1 WO 2015172706A1 CN 2015078766 W CN2015078766 W CN 2015078766W WO 2015172706 A1 WO2015172706 A1 WO 2015172706A1
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antigen
schistosoma mansoni
combination
seq
antigens
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PCT/CN2015/078766
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Chinese (zh)
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李明
徐新东
吴英松
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广州市达瑞生物技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to the field of biotechnology, and in particular, the present invention discloses a series of antigen combinations derived from Schistosoma mansoni with specific diagnostic effects and uses thereof.
  • Schistosoma mansoni is the most prevalent schistosomiasis. There are 54 countries with schistosomiasis in the world, including Africa, South America and Central Asia. The number of infected people exceeds 80 million. Compared with Schistosoma japonicum, the natural end-host of Schistosoma mansoni is mainly human and other primates, while the natural end-host of Schistosoma japonicum includes more than forty animals such as cattle and pigs and rodents. In addition, the intermediate host of Schistosoma mansoni is aquatic double umbilical snail, and the intermediate host of Schistosoma japonicum is amphibious snail. The difference between Schistosoma japonicum and Schistosoma mansoni is also manifested in the patient's condition, the former is more severe and the latter is lighter.
  • the lack of efficient, inexpensive and convenient schistosomiasis diagnostic techniques is one of the main factors leading to the difficulty in the prevention and treatment of schistosomiasis.
  • Accurate diagnostic techniques can be used not only for patient diagnosis, but also for classifying epidemic areas, assessing prevalence, and assessing prevention and control effects.
  • the pathogen detection methods mainly include the Kato-Katz Technique and the Hatching Test; the immunological detection methods mainly include Indirect Hemagglutination Assay (IHA) and Enzyme-linked immunosorbent assay (Enzyme).
  • ELISA -Linked Immunosorbent Assay
  • COPT Circum Oval Precipitating Test
  • DIGFA Dotimmunogold Filtration Assay
  • ELISA technology has the advantages of simple operation, high sensitivity, easy standardization, and is widely used in the diagnosis of various diseases, so it is the priority of schistosomiasis diagnosis.
  • the key to ELISA technology is the choice of antigen.
  • the antigen used in the diagnostic ELISA of schistosomiasis antibody on the market is soluble egg antigen (SEA), but SEA is a crude antigen with complex components, making the specificity of SEA-ELISA poor, prone to cross-reaction, and inability to distinguish between current infection and past infection.
  • SEA is usually extracted from eggs in the liver of animals, and production conditions are difficult to control, which results in higher SEA costs and instability between batches.
  • Recombinant proteins produced by genetic engineering have the advantages of high yield, low price, and low ELISA cross-reactivity. They are potential novel antigens to replace SEA, but recombinant protein ELISA The key is the screening of highly sensitive, highly specific antigen molecules.
  • Immunology is the interdisciplinary study of immunology and genomics. It uses immunological techniques to screen out a set of molecules that interact with the host's immune system from the genome, proteome, and transcriptome. We call this group an immunoome. Molecules in the immunization group have great potential value for the diagnosis of disease immunology. Therefore, immunohistochemical research is an important part of the research of genomics in the post-schistosomiasis era, and the results will directly guide the diagnosis and development of schistosomiasis. The vast and vast genomic data of schistosomiasis provides a powerful information platform for our comprehensive and in-depth study of schistosomiasis. How to screen out new targets for schistosomiasis, especially the diagnosis and control of Schistosoma mansoni, from the genome has become an urgent problem for researchers of schistosomiasis.
  • the inventors found 17 candidate diagnostic molecules in the EST database of Schistosoma mansoni (SmSP-001, SmSP-004, SmSP-013, SmSP-015, SmSP-016, SmSP-017, SmSP-019, SmSP-051, SmSP-080, SmSP-129, SmSP-144, SmSP-160, SmSP-162, SmSP-196, SmSP-203, SmSP-204, SmSP-216).
  • the present invention investigates the diagnostic effects of these 17 diagnostic molecules.
  • a combination of Schistosoma mansoni antigens comprising two or more antigens from the group consisting of:
  • the antigen in the antigen combination is directed against an antibody that is different.
  • the antigen combination comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 selected from the group consisting of SEQ ID NO .: polypeptide of 1-17.
  • the antigen combination comprises at least the polypeptide of SEQ ID NO.: 3.
  • the antigen combination comprises the polypeptide of SEQ ID NO.: 3, and at least one or more selected from the group consisting of SEQ ID NO.: 1, 2, 4, 5, 6, and 7. a polypeptide represented by 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.
  • the antigen combination comprises the polypeptide of SEQ ID NO.: 3, and the polypeptide of SEQ ID NO.: 12 and/or 17.
  • an isolated polynucleotide combination wherein the polynucleotides in the polynucleotide combination respectively encode an antigen in the antigen combination of the first aspect of the invention.
  • a vector comprising the polynucleotide combination of the second aspect of the invention.
  • the vector may further comprise the antigen of any one of SEQ ID NO.: 1-17.
  • the combination comprises two or more of the polynucleotides described.
  • the vector may also be a combination of vectors comprising a polynucleotide encoding the antigen of SEQ ID NO.: 1-17.
  • a genetically engineered host cell comprising the vector of the third aspect of the present invention or a chromosome integrated with the multinuclear of the polynucleotide combination of the second aspect of the present invention Glycosylate.
  • a method of preparing a combination of Schistosoma mansoni antigens comprising the steps of:
  • the method further comprises mixing the antigens obtained in (b) to prepare a Schistosoma mansoni antigen combination.
  • the antigen combination may be a mixture or a combination comprising a plurality of independent antigens.
  • the Schistosoma mansoni antigen or the antigen combination according to the first aspect of the present invention for the preparation of a reagent or a kit for diagnosing Schistosoma mansoni, wherein the Schistosoma mansoni antigen comprises a Or a plurality of polypeptides selected from the group consisting of SEQ ID NO.: 1-17.
  • kits for detecting Schistosoma mansoni comprising: a container or a carrier; and an antigen combination according to the first aspect of the invention located in or on the container
  • the detection reagents are constructed.
  • the kit further comprises an enzyme binding solution, a reaction substrate, and an optional instruction.
  • the kit further comprises a component selected from the group consisting of: a reaction stop solution, Sample diluent, and wash solution.
  • the reagent comprises a solid phase carrier and the antigen of any one of SEQ ID NO.: 1-17 or a combination thereof coated on the solid phase carrier, preferably, the present invention The antigen combination described on the one hand.
  • the kit or reagent is used to detect whether the sample to be tested is infected with Schistosoma mansoni.
  • the sample to be tested is from a population of endemic areas of Schistosoma mansoni.
  • the sample to be tested is from a bovine or other mammal, such as a human.
  • the invention also provides an antibody which specifically binds to one or more polypeptides selected from the group consisting of SEQ ID NO.: 1-17.
  • the antibody binds to the polypeptide of SEQ ID NO.: 3.
  • the antibody specifically binds to a polypeptide represented by SEQ ID NO.: 1, 3, 6, 8, 10, 12, 13, 15, or 17, respectively.
  • an antigen-antibody complex comprising:
  • polypeptides selected from the group consisting of SEQ ID NO.: 1-17;
  • the antigen-antibody complex comprises at least the polypeptide of SEQ ID NO.: 3 and an antibody that specifically binds to the polypeptide of SEQ ID NO.: 3.
  • the antigen-antibody complex for:
  • a method for detecting whether a sample is infected with Schistosoma mansoni in a diagnostic or non-diagnostic manner comprising the steps of:
  • step (3) if the sample in step (3) exhibits the antigen-antibody complex, it indicates that the sample is infected with Schistosoma mansoni.
  • amino acid sequence of the positive antigen is as shown in SEQ ID NOS: 1-17.
  • step (1) comprises: preparing a Schistosoma mansoni antigen represented by SEQ ID NO.: 3, and/or any one selected from the group consisting of SEQ ID NO.: 1, 2, 4, 5, 6
  • Figure 1 shows the R value of the expression level of the antigen of the present invention in normal and Schistosoma mansoni patients.
  • Figure 2 shows the PCR product of electrophoresis for strongly positive antigens.
  • FIG. 3 shows the SDS-PAGE detection of SmSP-013.
  • FIG. 4 shows the SDS-PAGE detection of SmSP-080.
  • Figure 5 shows SDS-PAGE detection of SmSP-160 and SmSP-216.
  • Schistosomiasis is a serious hazard to the health of humans and other mammals.
  • Schistosoma mansoni is the parasite that has the widest range of infections, the highest number of infections and threats, and the heaviest burden of disability-adjusted life years. It is currently popular in 54 countries around the world. The pathogenesis of acute schistosomiasis is still unclear. The treatment of Schistosoma mansoni can not achieve good control effects due to differences in regions and populations.
  • Immunology is an interdisciplinary study of immunology and genomics that uses immunological techniques to screen a set of molecules that interact with the host's immune system, ie, the immune group, from the genome, proteome, and transcriptome. Immunome).
  • the molecules in the immunization group have great potential value for the diagnosis of disease immunology, and are an important part of the research in the genome era after Schistosoma mansoni. How to select new targets for the diagnosis and prevention of schistosomiasis from the immunization group has become an urgent problem for researchers in the field.
  • Schistosoma mansoni is one of the six most infected human schistosomiasis, with the widest range of epidemics, the most infectious and threatened population, and the most severely burdened life-adjusted life-year parasite.
  • Mainly popular in Africa including Egypt, Sudan, Ethiopia, Kenya, Africa, Mozambique, Moscow, Zambia, Congo, etc.
  • South America Brazil, Guyana, Dominica, Caribbean, etc.
  • Asia Asia (Arabic).
  • Schistosoma mansoni is commonly prevalent in developing countries with poor sanitation and in poor countries, and human feces containing eggs are the main source of infection.
  • the susceptible population is dominated by fishermen, farmers and children.
  • residents of endemic areas have partial immunity due to repeated infections. First-time infection of residents in non-endemic areas can cause acute schistosomiasis.
  • an antigen is a substance that stimulates the body to produce a (specific) immune response and binds to immune response product antibodies and sensitized lymphocytes in vitro to produce an immune effect (specific reaction).
  • An antigen is a substance that stimulates the body to produce a (specific) immune response and binds to immune response product antibodies and sensitized lymphocytes in vitro to produce an immune effect (specific reaction).
  • an antigen There are two basic properties of an antigen, one is the ability to induce an immune response, that is, immunogenicity, and the other is to react with the product of an immune response, that is, antigenicity.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and proteins in the natural state in living cells are not isolated and purified, but the same polynucleotide or protein is separated from other substances existing in the natural state, and is isolated and purified.
  • isolated positive antigen is meant that the antigen is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify positive antigens using standard protein purification techniques. A substantially pure protein antigen produces a single major band on a non-reducing polyacrylamide gel. The purity of the antigen can be analyzed by amino acid sequence.
  • the antigen of the present invention may be a naturally purified product, or a chemically synthesized product, or may be produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Health.
  • the antigens of the invention may be glycosylated and non-glycosylated according to the host used in the recombinant production protocol. Antigens of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the antigen.
  • fragment refers to an antigen that substantially retains the same biological function or activity of the native antigen of the invention.
  • the antigenic fragment, derivative or analog of the present invention may be (i) an antigen having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) an antigen having a substituent group in one or more amino acid residues, or (iii) an antigen and another compound (such as a compound that prolongs the half-life of the antigen, such as poly Ethylene glycol) is formed by fusing the formed antigen, or (iv) an additional amino acid sequence is fused to the antigen sequence.
  • fragments, derivatives and analogs are within the purview of those skilled in the art.
  • the term "antigen" also includes variant forms having the same function. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • amino acids usually within 20 preferably within 10, more preferably within 5
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • Modifications include chemically derived forms of the antigen, such as acetylation, carboxylation, glycosylation, in vivo or in vitro. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine.
  • a preferred class of active derivatives means up to 5, preferably up to 3, more preferably up to 2, optimally 1 amino acid is similar in nature to the sequence of SEQ ID NO.: 1-17 A similar amino acid is replaced to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp
  • SEQ ID NO. Antigen name 1 SmSP-001 2 SmSP-004 3 SmSP-013 4 SmSP-015 5 SmSP-016 6 SmSP-017 7 SmSP-019 8 SmSP-051 9 SmSP-080 10 SmSP-129 11 SmSP-144 12 SmSP-160 13 SmSP-162 14 SmSP-196 15 SmSP-203 16 SmSP-204 17 SmSP-216
  • antigen combination refers to the diagnosis or pairing of the Schistosoma mansoni positive antigen found in the present invention. For the purpose, carry out the required combination of forms. For example, combining two or more antigens selected from the polypeptides of SEQ ID NOS.: 1-17 or derived polypeptides thereof can improve the diagnostic specificity and positive diagnostic rate of Schistosoma mansoni.
  • the antigen combination of the present invention comprises at least the polypeptide represented by SEQ ID NO.: 3, and the other one or more selected from the group consisting of SEQ ID NO.: 1, 2, 4, 5, 6, 7, 8, The antigens shown in 9, 10, 11, 12, 13, 14, 15, 16, and 17. Experiments have shown that Schistosoma mansoni can be detected more efficiently using an antigen combination comprising at least the polypeptide of SEQ ID NO.: 3.
  • conventional genetic engineering methods can be employed, such as introducing a coding sequence of one or several antigens of the present invention into a vector, and introducing into a host cell, or introducing only one antigen of the present invention into the vector.
  • sequences, which form a combination of vectors, are introduced together into a host cell, and such methods are well known to those skilled in the art.
  • 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 can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • polynucleotide encoding an antigen of the present invention may be a polynucleotide comprising the antigen, or a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • the invention also relates to variants of the above polynucleotides. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • the nucleotide sequence of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the DNA sequence encoding the antigen of the present invention (or a fragment thereof, or a derivative thereof) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Chemical synthesis Mutations are introduced into the antigenic sequences of the invention.
  • a 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 for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the preparation method of the antigen of the present invention has the following steps:
  • a polynucleotide sequence can be inserted into a recombinant expression vector.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing antigen-encoding DNA sequences and appropriate transcription/translation control signals.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the term "vector,” as used herein, includes a carrier combination of the vector itself and a plurality of vectors, i.e., the vector may contain a polynucleoside encoding one or more antigens selected from the sequences set forth in SEQ ID NO.: 1-17. acid.
  • the vector contains a polynucleotide encoding an antigen selected from the sequences set forth in SEQ ID NO.: 1-17, and these vector combinations containing a polynucleotide encoding one sequence are capable of forming a vector combination. It is also possible to have a vector comprising two or more polynucleotides selected from the sequences shown in SEQ ID NO.: 1-17.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E.
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art. Another method is to use MgCl 2. Conversion can also be carried out by electroporation if desired.
  • DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the antigenic protein of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the antigenic protein can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If necessary, profitable
  • the antigenic proteins are separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention is based on the method for expressing glutathione-S-transferase (GST) fusion protein for screening high-throughput protein antigens, comprising the steps of: (1) predicting the expression sequence tag (EST) of Schistosoma mansoni using bioinformatics methods;
  • the database is preferably a protein sequence of NCBI, the prediction software preferably SignalP 3.0 ( http://www.cbs.dtu.dk/services/SignalP/ ); (2) amplification
  • the target gene corresponding to each signal peptide protein is cloned into an expression vector, preferably the expression vector is a GST expression vector, and the Escherichia coli is induced to induce the expression of the antibody-GST fusion protein; (3) the fusion protein is placed in the chelate GST fusion protein is captured in the wells of the GSH-filled multi-well plate; (4) The serum containing the Schistosoma mansoni antibody is placed in a multi-well plate for
  • kits for detecting Schistosoma mansoni comprises the following components: a container or carrier; and the positive antigen of the first aspect of the invention located in or on the container.
  • the kit further includes an enzyme binding solution, a reaction substrate, and an optional instructions.
  • the kit may further comprise a component selected from the group consisting of a reaction stop solution, a sample diluent, and a wash solution.
  • a preferred kit for detecting Schistosoma mansoni includes: a container and a coating solution in the container, a horseradish peroxidase-labeled mouse anti-human IgG (H+L) antibody, a substrate solution TMB, and a thinner Concentrated sulfuric acid reaction stop solution and sample diluent. More preferably, blank controls, positive and negative controls are also included and monitored to see if the test process meets the criteria.
  • the antigen has high sensitivity and is not affected by the degree of infection of the test sample. It can still be detected in the case of EPG ⁇ 10, and the sensitivity is high.
  • the antigen detection of the sample of the invention has a false positive rate which is far lower than the commercially available SEA method.
  • the antigen can be read using a common ELISA method.
  • the expression of the target protein was induced at 1 mmol/L, 37 ° C, and 250 rpm; the expression was stopped for 6 hours, and the culture was stopped; the cells were collected and stored at -80 ° C for use.
  • Dissolution of inclusion bodies freeze and thaw the cells twice; add 300 ⁇ l of B-PER (addition of DNase, RNase, PMSF) to lyse the cells, mix by pipetting, transfer to EP tube; centrifuge at room temperature for 1 min at 13,000 rpm; remove The supernatant was rinsed into a new Ep tube, and stored as a inclusion body by freezing.
  • B-PER addition of DNase, RNase, PMSF
  • the inclusion body was washed with PBS containing 1% Triton-X100 as a washing solution, and mixed, centrifuged at 13,000 rpm for 5 min, and the supernatant was discarded; Repeat the washing step, centrifuge again to discard the supernatant; dissolve the inclusion body with the inclusion body solution, and obtain about 0.1 g of inclusion body precipitate in 1 ml of induced bacterial solution, add 1 ml of the solution, mix by blowing, shake at room temperature overnight, and fully dissolve the inclusion body. .
  • Inclusion body renaturation The dissolved solution was centrifuged at 13,000 rpm for 5 min at room temperature, and the supernatant was taken; in a 96-well deep-well culture plate, 50 ⁇ l of the denatured solution and 1 ml of the reconstituted solution were added to each well; Overnight; collect the supernatant by centrifugation.
  • Serum adsorption The pGEX-4T-1 plasmid was transformed into Escherichia coli B21 (DE3), and 100 ml of GST expression broth and B21 broth were prepared according to the previous expression method. The cells were collected by centrifugation and weighed with 10 ml PBS. After suspension and sonication, the supernatant was collected for use; 1 ml column volume GSH packing, 3 ml GST expression supernatant and 9 ml PBS were mixed, mixed at room temperature for 1 h, centrifuged at 1000 rpm to collect the precipitate, and washed with 10 ml PBS five times, at which time the filler was combined with GST.
  • pET28(a) expression vector A strong positive antigen gene was digested from recombinant pGEX-4T-1, ligated into pET28(a) vector, and positive clones were screened by Kan + plate.
  • Bacterial cell lysis collect bacteria, freeze and thaw the bacteria twice; lyse the bacteria after freezing and thawing twice with 10ml bacterial protein extraction reagent B-PER (addition of DNase, RNase, PMSF, etc.) to lyse the bacteria and transfer to the beaker Medium, stirring slowly for 30 min, sonicating for 10 min.
  • B-PER additional of DNase, RNase, PMSF, etc.
  • Protein renaturation The purified protein solution was dialyzed in a refolding solution at a volume ratio of 1:10, and the fresh reconstituted solution was changed every 4 hours at 4 ° C for 3 times; Protein in a volume ratio of 1:10, inclusive 10% glycerol was dialyzed in PBS, and fresh dialysate was changed every 5 hours at 4 ° C for 3 times; SDS-PAGE; concentrated protein solution was concentrated to about 1 ml to determine the protein concentration.
  • Solid phase carrier coated with antigen The protein antigen was coated in a polystyrene reaction well, and the coating solution formulation: 1.5 g of Na 2 CO 3 and NaHCO 3 was dissolved in 1000 ml of distilled water.
  • TMB A solution (3.2 ⁇ l of 1.5% H 2 O 2 per ml of 0.005% sodium acetate-citrate buffer, stored at 4 ° C protected from light); TMB B solution (TMB 0.08 g, After adding 40 ml of DMSO to dissolve, add 60 ml of methanol, mix and add 100 ml of 0.005% sodium acetate-citrate buffer, shake for 1 hour in the dark, stand at room temperature for 3 hours, store at 4 ° C in the dark;) before use, liquid A and B The liquid is mixed in equal volume.
  • Sample dilution Dilute the test serum by 1:100 with the sample diluent.
  • Sample addition reaction 100 ⁇ l of diluted serum samples were added to each well of the sample test well, and 2 replicate wells were detected in parallel for each sample. At the same time, positive, negative and blank control 2 duplicate wells were taken, 100 ⁇ l of each positive and negative control substance were added to the well, and the blank control was only added with the sample dilution. Incubate at 37 ° C for 60 minutes in the dark.
  • Washing ⁇ dry the liquid in the well, fill each well with the washing solution, let stand for 2 minutes, dry, repeat the washing 4 times, and pat the last time.
  • HRPase horseradish peroxidase
  • Reading and data processing zero adjustment with blank control, read OD value at wavelength 450nm; OD value of the hole to be tested is greater than or equal to 2.1 times of the negative control is positive, when the negative control OD value is less than 0.05, calculate by 0.05 .
  • the serum and normal human serum of Schistosoma mansoni patients were collected to compare the diagnostic specificity and sensitivity of single antigens and different combinations of antigens. The results are shown in Table 6. The specificity of the antigen combination was not significantly different from that of the single antigen, but the sensitivity was superior to that of the single antigen.

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Abstract

L'invention concerne le criblage d'antigènes de diagnostic de Schistosoma mansoni et une utilisation associée. L'invention concerne en particulier des antigènes provenant de Schistosoma mansoni, et l'association antigénique comprenant deux antigènes ou plus choisis dans le groupe : (a) des polypeptides illustrés en SEQ ID NO : 1 -17 ; (b) des polypeptides dérivés ayant une immunogénicité obtenue par substitution, délétion ou addition d'un ou plusieurs résidus d'acides aminés des séquences d'acides aminés SEQ ID NO : 1 -17 ; (c) des polypeptides ayant une immunogénicité, présentant une homologie ≥ 90 % avec les séquences d'acides aminés illustrées en SEQ ID NO : 1 -17 ; ou (d) le fragment des polypeptides (a)-(c) ayant une immunogénicité, les antigènes dans la combinaison antigénique étant dirigés contre différents anticorps. La sérologie positive à Schistosomiasis mansoni au moyen de ces antigènes indique leur grande sensibilité et leur grande spécificité. L'invention concerne également une méthode et un kit permettant de diagnostiquer et de surveiller la bilharziose.
PCT/CN2015/078766 2014-05-12 2015-05-12 Criblage d'antigènes de diagnostic de schistosoma mansoni et utilisation associée WO2015172706A1 (fr)

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PROTASIO, A.V. ET AL.: "A Systematically Improved High Quality Genome and Transcriptome of the Human Blood Fluke Schistosoma mansoni''.", PLOS NEGLECTED TROPICAL DISEASES, vol. 6, no. 1, 10 January 2012 (2012-01-10), pages e1455, XP055236158 *

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