WO2015172706A1 - Screen of schistosoma mansoni diagnostic antigen and use - Google Patents

Abstract

Disclosed are screen of Schistosoma mansoni diagnostic antigens and a use thereof. In particular, the antigens are from Schistosoma mansoni, and the antigen combination comprises two or more antigens selected from the group of: (a) polypeptides shown as SEQ ID NOs:1-17; (b) derived polypeptides having immunogenicity formed by one or more amino acid residue(s) substitutions, deletions or additions from the amino acid sequences of SEQ ID NOs:1-17; (c) polypeptides having immunogenicity, with ≥90% homology with the amino acid sequences shown as SEQ ID NOs:1-17; or (d) the fragment of polypeptides (a) - (c) having immunogenicity, wherein antigens in the antigen combination are directed at different antibodies. The Schistosomiasis mansoni serologic detection indicates that using these positive antigens for detecting Schistosoma mansoni have a high sensitivity and specificity. Also provided in the present invention are a method and a kit for correspondingly diagnosing and monitoring the Schistosomiasis mansoni disease.

Description

Screening and application of diagnostic antigens of Schistosoma mansoni Technical field

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.

Background technique

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. At present, there are three main methods for diagnosing schistosomiasis, one is pathogen detection method, the other is immunological detection method, and the third is molecular biological detection method. 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). -Linked Immunosorbent Assay (ELISA), Circum Oval Precipitating Test (COPT) and Dotimmunogold Filtration Assay (DIGFA); molecular biology methods are diagnosed by schistosomiasis-specific nucleic acid amplification technology . Each of the above methods has advantages and disadvantages. The results of the pathogen test are still the gold standard for the diagnosis of schistosomiasis, but the sensitivity of the pathogen detection is low, time-consuming and laborious, and there is also the danger of environmental pollution. The immunological methods of COPT and IHA are highly specific, but have low sensitivity and are not widely used. The molecular biology method has high sensitivity, but it is prone to false positives. At the same time, the method requires high experimental conditions, and currently only stays in the laboratory research stage. In contrast, 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. At present, 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. In addition, 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.

In order to screen new diagnostic targets, in 1994, the World Health Organization (WHO) launched the Schistosomiasis Genome Project. In recent years, great progress has been made in the study of schistosomiasis, and the genome sketches of Schistosoma mansoni and Schistosoma japonicum have been drawn, and more than 10,000 genes have been annotated. At the same time, the EST database and proteome database have also been established. These important databases can be used not only to study the growth and development mechanisms of schistosomiasis at the molecular level, to explore the mechanism of schistosomiasis-host interaction, but more importantly, to serve new methods for the prevention and control of schistosomiasis, to screen new diagnostic molecules, candidate vaccine molecules and New targets for drugs, etc. After the identification of a large number of schistosomiasis genes and proteins, people have been unable to satisfy the biological functions of genes at a single gene level, and schistosomiasis research has entered the era of "omics". However, there is currently no way to clarify which molecules are the targets we need directly from the schistosomiasis genome, proteome or transcriptome. Therefore, there is an urgent need for new technologies to combine omics data with specific research areas. 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.

Summary of the invention

It is an object of the present invention to provide a combination of positive antigens for Schistosoma mansoni and uses thereof.

Specifically, 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.

In a first aspect of the invention, there is provided a combination of Schistosoma mansoni antigens comprising two or more antigens from the group consisting of:

(a) a polypeptide as shown in SEQ ID NO.: 1-17;

(b) an immunogenic derivative polypeptide formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 1-17 with one or more amino acid residues;

(c) an immunogenic polypeptide having a homology of ≥90% to the amino acid sequence shown in SEQ ID NOS: 1-17;

(d) a fragment of the immunogenic polypeptides (a)-(c);

Wherein the antigen in the antigen combination is directed against an antibody that is different.

In another preferred embodiment, 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.

In another preferred embodiment, the antigen combination comprises at least the polypeptide of SEQ ID NO.: 3.

In another preferred embodiment, 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.

In another preferred embodiment, the antigen combination comprises the polypeptide of SEQ ID NO.: 3, and the polypeptide of SEQ ID NO.: 12 and/or 17.

In a second aspect of the invention, there is provided 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.

In a third aspect of the invention, there is provided a vector comprising the polynucleotide combination of the second aspect of the invention.

In another preferred embodiment, the vector may further comprise the antigen of any one of SEQ ID NO.: 1-17.

In another preferred embodiment, the combination comprises two or more of the polynucleotides described.

In another preferred embodiment, the vector may also be a combination of vectors comprising a polynucleotide encoding the antigen of SEQ ID NO.: 1-17.

According to a fourth aspect of the present invention, 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.

According to a fifth aspect of the invention, there is provided a method of preparing a combination of Schistosoma mansoni antigens, the method comprising the steps of:

(a) cultivating the host cell of the fourth aspect of the invention under conditions suitable for expression;

(b) Isolate the Schistosoma mansoni positive protein antigen from the culture.

In another preferred embodiment, the method further comprises mixing the antigens obtained in (b) to prepare a Schistosoma mansoni antigen combination.

In another preferred embodiment, the antigen combination may be a mixture or a combination comprising a plurality of independent antigens.

According to a sixth aspect of the present invention, there is provided the use of 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.

According to a seventh aspect of the invention, there is provided a kit for detecting Schistosoma mansoni, the kit 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.

In another preferred embodiment, the kit further comprises an enzyme binding solution, a reaction substrate, and an optional instruction. Preferably, the kit further comprises a component selected from the group consisting of: a reaction stop solution, Sample diluent, and wash solution.

In another preferred embodiment, 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.

In another preferred embodiment, the kit or reagent is used to detect whether the sample to be tested is infected with Schistosoma mansoni.

In another preferred embodiment, the sample to be tested is from a population of endemic areas of Schistosoma mansoni.

In another preferred embodiment, the sample to be tested is from a bovine or other mammal, such as a human.

In another preferred embodiment, 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.

In another preferred embodiment, the antibody binds to the polypeptide of SEQ ID NO.: 3.

In another preferred embodiment, the antibody specifically binds to a polypeptide represented by SEQ ID NO.: 1, 3, 6, 8, 10, 12, 13, 15, or 17, respectively.

According to an eighth aspect of the present invention, an antigen-antibody complex is provided, the complex comprising:

(i) one or more polypeptides selected from the group consisting of SEQ ID NO.: 1-17;

(ii) an antibody that specifically binds to the polypeptide in (ii).

In another preferred embodiment, 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.

In a ninth aspect of the invention, there is provided the use of the antigen-antibody complex, the complex for:

(a) preparing a reagent or kit for detecting Schistosoma mansoni; and

(b) Used as a positive control for the detection of Schistosoma mansoni.

According to a tenth aspect of the present invention, there is provided a method for detecting whether a sample is infected with Schistosoma mansoni in a diagnostic or non-diagnostic manner, comprising the steps of:

(1) preparing a Schistosoma mansoni antigen or an antigen combination thereof as shown in any one of SEQ ID NO.: 1-17;

(2) contacting the antigen obtained in the step (1) or an antigen combination thereof with the sample to be detected;

(3) detecting whether the antigen-antibody complex of the ninth aspect of the invention is contained in the sample;

Wherein, if the sample in step (3) exhibits the antigen-antibody complex, it indicates that the sample is infected with Schistosoma mansoni.

In another preferred embodiment, the amino acid sequence of the positive antigen is as shown in SEQ ID NOS: 1-17.

In another preferred embodiment, 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 The Schistosoma mansoni antigen shown in 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

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.

Figure 3 shows the SDS-PAGE detection of SmSP-013.

Figure 4 shows the SDS-PAGE detection of SmSP-080.

Figure 5 shows SDS-PAGE detection of SmSP-160 and SmSP-216.

detailed description

Through extensive and intensive research, the present inventors have for the first time accidentally screened 17 positive antigens that can be recognized by the sera of Schistosoma mansoni in Schistosoma mansoni, of which 5-10 are strongly positive antigens. The experimental results demonstrate that these antigen combinations, especially the antigen SmSP-013 or its combination with other antigens, have high sensitivity and specificity for detecting Schistosoma mansoni. Based on the present invention, it can be used to prepare a reagent or kit for diagnosing and detecting Schistosoma mansoni. On the basis of this, the present invention has been completed.

Schistosomiasis and its omics research

Schistosomiasis is a serious hazard to the health of humans and other mammals. Among them, 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.

In order to screen new diagnostic and therapeutic targets, the World Health Organization (WHO) launched the Schistosomiasis Genome Project in 1994, and in recent years, the research on Schistosoma mansoni has made great progress, and the genome sketch of Schistosoma mansoni has been drawn, and More than 10,000 genes have been annotated, and EST databases and proteomic databases have been established. These important databases can be used not only to study the growth and development mechanisms of Schistosoma mansoni at the molecular level, but also to explore the mechanism of Schistosoma mansoni-host interaction and, more importantly, to serve Schistosoma japonicum. Research on new methods of disease prevention and control, screening of new diagnostic molecules, candidate vaccine molecules and new targets for drugs. However, there is currently no way to clarify which molecules are the targets we need directly from the genome, proteome or transcriptome of Schistosoma mansoni. Therefore, there is an urgent need for new technologies to combine omics data with specific research areas. Immunology (immunomics) 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

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, Tanzania, Mozambique, Zimbabwe, Zambia, Congo, etc.), South America (Brazil, Guyana, Dominica, Caribbean, etc.), 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.

Positive antigen

In the present invention, the terms "antigen" or "positive antigen" are used interchangeably and refer to a protein or polypeptide which is capable of specifically binding to a Schistosoma mansoni antibody. 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). 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.

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, 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. . By "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. As used herein, the terms "fragment," "derivative," and "analog" refer 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. These fragments, derivatives and analogs are within the purview of those skilled in the art.

In the present invention, 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. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, 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 (usually without altering the primary structure) 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.

Table 1

Initial residue	Representative substitution	Preferred 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

Gly(G)	Pro; Ala	Ala
			His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
			Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

The antigen names and corresponding serial numbers shown in SEQ ID NO.: 1-17 of the present invention are shown in Table 2:

Table 2

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

The term "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.

Usually, a variety of positive antigens can be selected for combination to improve the diagnosis rate. Of course, a few strong positive antigens can also be selected for combination, and a wide range of screening can be performed with the most economical choice. Preferably, 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.

In order to prepare the antigen combination of the present invention, 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. The 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.

Antigen coding sequence

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. The term "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. For PCR amplification, 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. Once the relevant sequences are obtained, 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. In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, 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.

Antigen preparation method

The preparation method of the antigen of the present invention has the following steps:

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

(2) a host cell cultured in a suitable medium;

(3) Separating and purifying the antigen of the present invention from a culture medium or a cell.

In the present invention, 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. Preferably, 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. 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. When the host is a prokaryote such as E. coli, 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. When the host is a eukaryote, the following 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. After the host cell has grown to the appropriate cell density, 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.

Antigen screening method

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; In a preferred embodiment of the invention, 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 antigen-antibody detection, and the pores forming the specific antigen-antibody complex are screened. The corresponding candidate protein is the Schistosoma mansoni positive antigen.

Kit

The invention also provides a kit for detecting Schistosoma mansoni. In general, the kit of the present invention 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. In another preferred embodiment, the kit further includes an enzyme binding solution, a reaction substrate, and an optional instructions. Preferably, 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 main advantages of the invention are:

1. 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.

2. High specificity, the antigen detection of the sample of the invention has a false positive rate which is far lower than the commercially available SEA method.

3. Easy to operate, the antigen can be read using a common ELISA method.

4. The results are stable and highly reproducible.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press), or as recommended by the manufacturer. conditions of. Percentages and fractions are by weight unless otherwise stated.

Example 1 Screening of important antigens of Schistosoma mansoni

1. Predict the secreted protein of Schistosoma mansoni: download the EST database of Schistosoma mansoni from NCBI, and the corresponding protein sequence; introduce the protein sequence into the signal peptide online prediction software SignalP 3.0 (http://www.cbs.dtu.dk/services /SignalP/) is predicted; based on the predicted results, the sequence predicted to be a signal peptide protein is selected according to a p-value greater than 0.1.

2. Gene cloning: extraction of total RNA from Schistosoma mansoni; amplification of the target gene by RT-PCR, recovery of the target fragment, restriction enzyme digestion, cloning of the target fragment into the commercially available expression vector pGEX-4T-1; transformation of commercially available large intestine Bacillus Top10, positive clones were sequenced to confirm the correct sequence.

3. Recombinant protein-induced expression: extract the plasmid, transform the pGEX-4T-1 recombinant plasmid into the commercially available Escherichia coli B21; pick a single clone, inoculate a 96-well bacterial deep-well culture plate, incubate at 37 ° C, 250 rpm overnight, press 1 Percentage of 96-well bacterial deep-well culture plates containing 1 ml of 2×YT (containing 100 μg/ml Amp) liquid medium per well, incubated at 37 ° C for 250 rpm for about three hours to OD ₆₀₀ =0.4-1.0; IPTG was added to the final concentration. 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.

4. Dissolution of inclusion bodies: freeze and thaw the cells twice; add 300 μl 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. 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. .

5. 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 μl of the denatured solution and 1 ml of the reconstituted solution were added to each well; Overnight; collect the supernatant by centrifugation.

6. 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. Protein; resuspend GST/filler in 10ml PBS, dispense 1.9ml per tube; add 100μl patient serum to the dispensing tube, mix at room temperature for 5h, this step is to adsorb anti-GST antibody in serum; centrifuge at 1000rpm for 5min, remove the filler The supernatant was collected at a time when the serum dilution was 1:20; 3 ml of Bl21 (DE3) lysed supernatant was added to 2 ml of GST-adsorbed serum and mixed at room temperature for 5 h. This step is to adsorb antibodies against bacterial proteins. The serum dilution was 1:50; centrifuged at 13,000 rpm for 10 min, 500 μl of each tube was dispensed, and stored frozen for use.

7. Screening of seropositive clones: In a GSH-microplate, add 20 μl of refolding protein solution and 80 μl of PBS per well, and incubate overnight at 4 °C. At the same time, GST-expressing bacterial solution was used as a negative control, and PBS was used as a blank control. After the positive protein, this clone was used as a positive control.

Wash PBST (PBS + Tween) five times; 200μl 5% milk powder / PBST, blocking at room temperature for 2h; PBST wash five times; adsorbed serum diluted 1:20 with 5% milk powder, the final serum dilution is 1:1000; 100 μl/well, binding at 37 ° C for 1 h; washing with PBST five times; diluting HRP-labeled mouse anti-human IgG (H+L) antibody at 1:20,000, 100 μl/well, binding at 37 ° C for 1 h; washing with PBST 5 times; 100 μl of commercial Super Signal ELISA Femto was used to detect the fluorescence intensity value at 425 nm; calculate the ratio R of the fluorescence intensity of the sample and the negative control, and judge whether it is a positive protein according to the R size, and if R≥2, it is judged to be positive. The formula for calculating R is as follows:

The results are shown in Figure 1. Comparing the levels of specific antibodies in the serum of normal and Schistosoma mansoni infected patients, SmSP-001, SmSP-004, SmSP-013, SmSP-017, SmSP-019, SmSP-080, SmSP-160 The specific antibody levels in patients with SmSP-162, SmSP-196, SmSP-203, SmSP-216 and other antigens are higher than those in normal subjects, including SmSP-013, SmSP-080, SmSP-162 and SmSP-216. Antibody levels were significantly different between the two groups of serum.

Comparison of Diagnostic Sensitivity of Strong Positive Clones in Example 2

Strong positive antigens were selected and adsorbed onto GSH-microplates. GST (glutathione-S-transferase)-expressing bacterial solution was used as a negative control, PBS was used as a blank control; PBST was washed five times; 200 μl 5% milk powder /PBST, blocked for 2 h at room temperature; washed five times with PBST; the adsorbed serum was diluted with 5% milk powder at 1:20, and the final serum dilution was 1:1000; 100 μl/well, 1 h at 37 ° C; five washes with PBST; HRP-labeled mouse anti-human IgG (H+L) antibody was diluted 1:200, 100 μl/well, 1 h at 37 °C; PBST wash 5 The test was performed by adding 100 μl of a commercial Super Signal ELISA Femto, and the fluorescence intensity value was read at 425 nm; the R value was calculated. The results are shown in Table 3. The positive antigens of the present invention all have certain sensitivity.

Table 3: Comparison of diagnostic sensitivity of strongly positive clones

Example 3 Expression and Purification of Strong Positive Antigens

1. Construction of 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.

2. Expression of strong positive antigen protein: The recombinant pET28(a) vector was transformed into B21 (DE3) expression strain, and the positive clone was picked to prepare seed liquid, which was inoculated into the Erlenmeyer flask at 1%, and cultured at 37 ° C for 250 hours in a shake flask; Expression was induced by IPTG, and the final concentration of IPTG was 1 mM, and incubation was continued at 37 ° C and 250 rpm for 5 h.

3. 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.

4. Treatment of inclusion bodies: washing inclusion bodies: centrifugation at 15000 rpm for 10 min, collecting precipitates; resuspending the precipitate with 100 ml inclusion body washing solution: firstly disperse the precipitate, transfer to a beaker, then ultrasonically 1-2 min, thoroughly disperse the precipitate. Stir slowly for 30 min (washing solution formulation: 0.5 M urea, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl, 0.5% Triton-X100, pH = 8.0).

Dissolve inclusion body: Dissolve the precipitate with 100ml inclusion body solution: ultrasonic 1-2min, thoroughly disperse the precipitate, slowly stir for 30min; centrifuge at 15000rpm for 10min, collect the supernatant (solution formula: 8M urea, 0.1M NaH ₂ PO ₄ , 0.01 M Tris-HCl, pH = 8.0).

5. Ni column purification: Ni column is equilibrated with the solution; according to 1 ml column volume corresponding to 10 ml of protein solution, the two are mixed, 200 rpm, and incubated at room temperature for 30 min; the mixture is applied to the column to collect the post-column liquid; wash with 10 column volumes Wash the liquid, collect the washing solution (Ni column washing solution formula: 8 M urea, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl, 10 mM imidazole, pH=8.0); elute with 10 column volume eluent: collection wash Deliquoring, 1 ml/tube (Ni column eluent formulation: 8 M urea, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl, 500 mM imidazole, pH=8.0); SDS-PAGE was used to detect protein purity in each collection tube .

6. 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.

The results are shown in Figures 2, 3, 4 and 5. The results showed that the four antigens of SmSP-013, SmSP-080, SmSP-162 and SmSP-216 were successfully amplified (Fig. 2), and all four proteins were successfully expressed, and refined proteins were obtained (Fig. 3, 4, 5). ).

Example 4 Preparation of schistosomiasis ELISA kit

1. 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.

2. Preparation of enzyme binding solution HRP-labeled mouse anti-human IgG (H+L) antibody (Promega) was used.

3. Preparation of enzyme substrate solution: TMB A solution (3.2 μl 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.

4. Preparation of enzyme reaction stop solution: 100 ml of concentrated sulfuric acid was slowly added dropwise to 600 ml of ddH ₂ O, and the mixture was continuously stirred to a volume of 900 ml.

5. Prepare sample dilution: 5% milk powder / PBST.

6. Preparation of washing solution: PBST.

Example 5 detection method and operating procedure

1. Sample dilution: Dilute the test serum by 1:100 with the sample diluent.

2. Sample addition reaction: 100 μl 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 μl 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.

3. Washing: 液体 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.

4. Addition of enzyme: 100 μl of horseradish peroxidase (HRPase)-labeled mouse anti-human IgG (H+L) antibody was added to each well, and incubated at 37 ° C for 1 hour in the dark, washed as above, and patted dry.

5. Color development: Add 100 μl of enzyme substrate color solution to each well, incubate at 37 ° C for 10 minutes in the dark; add 50 μl of stop solution and immediately test with a microplate reader.

6. 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 .

Example 6 kit specificity

The normal person was collected and the false positive rate of the kit (positive antigen-ELISA) in the normal population was evaluated. The results showed that the positive antigen-ELISA had a better specificity than the SEA-ELISA. The results are shown in Table 4:

Table 4: Positive antigen - ELISA kit specificity

Example 7 kit sensitivity

Sample collection: The sera of patients (125) who had been diagnosed with Schistosoma mansoni were collected and evaluated for sensitivity to positive antigen-ELISA. The results are shown in Table 5:

Table 5: Sensitivity of positive antigen-ELISA kit

A large number of previous studies have shown that the sensitivity of SEA-ELISA is about 80%-95%. The conclusion of this study is basically consistent with the previous research conclusions. The sensitivity of the positive antigen-ELISA was consistent or better than that of the SEA-ELISA. In addition, because SEA is a mixed antigen, it is difficult to achieve accurate quality control, so there will always be some errors between batches, and the positive antigen protein is a recombinant antigen, which can achieve accurate quality control and reduce batch-to-batch variation. .

Example 8 Specificity and sensitivity of antigen combinations

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.

Table 6: Positive antigen combination - ELISA kit sensitivity

As a result, it was found that the antigen containing SEQ ID NO.: 3 and various other sequence combinations had higher specificity, and the antigen group positive rate of SEQ ID NO.: 3, 12, 13, and 17 reached 89.6%.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (11)

1. A combination of Schistosoma mansoni antigens, wherein the antigen combination comprises two or more antigens from the group consisting of:

   (a) a polypeptide as shown in SEQ ID NO.: 1-17;

   (b) an immunogenic derivative polypeptide formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 1-17 with one or more amino acid residues;

   (c) an immunogenic polypeptide having a homology of ≥90% to the amino acid sequence shown in SEQ ID NOS: 1-17;

   (d) a fragment of the immunogenic polypeptides (a)-(c);

   Wherein the antigen in the antigen combination is directed against an antibody that is different.
2. The antigen combination according to claim 1, wherein the antigen combination comprises at least the polypeptide of SEQ ID NO.: 3.
3. An isolated polynucleotide combination, wherein the polynucleotides in the polynucleotide combination encode the antigens in the antigen combination of claim 1.
4. A vector comprising the polynucleotide combination of claim 3.
5. A genetically engineered host cell, characterized in that the host cell comprises the vector of claim 4 or a polynucleotide in which the polynucleotide of claim 3 is integrated.
6. A method of preparing a combination of Schistosoma mansoni antigens, the method comprising the steps of:

   (a) cultivating the host cell of claim 5 under conditions suitable for expression;

   (b) Isolate the Schistosoma mansoni positive protein antigen from the culture.
7. Use of the Schistosoma mansoni antigen or the antigen combination according to claim 1, characterized in that the reagent or kit for preparing a diagnosis of Schistosoma mansoni, wherein the Schistosoma mansoni antigen comprises one or more selected from the group consisting of SEQ ID NO.: The polypeptide of 1-17.
8. A kit for detecting Schistosoma mansoni, characterized in that the kit comprises: a container or a carrier; and a detection reagent comprising the antigen combination according to claim 1 located in the container or on the carrier.
9. An antigen-antibody complex characterized in that the complex comprises:

   (i) one or more polypeptides selected from the group consisting of SEQ ID NO.: 1-17;

   (ii) an antibody that specifically binds to the polypeptide in (ii).
10. Use of the antigen-antibody complex of claim 10, wherein the complex is for:

    (a) preparing a reagent or kit for detecting Schistosoma mansoni; and

    (b) Used as a positive control for the detection of Schistosoma mansoni.
11. A method for detecting whether a sample is infected with Schistosoma mansoni in a diagnostic or non-diagnostic test, comprising the steps of:

    (1) preparing a Schistosoma mansoni antigen or an antigen combination thereof as shown in any one of SEQ ID NO.: 1-17;

    (2) contacting the antigen obtained in the step (1) or an antigen combination thereof with the sample to be detected;

    (3) detecting whether the antigen-antibody complex of claim 10 is contained in the sample;

    Wherein, if the sample in step (3) exhibits the antigen-antibody complex, it indicates that the sample is infected with Schistosoma mansoni.

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