CN1683405A - Clonorchis sinensis class II 14-3-3 protein, its coding nucleic acid and its application - Google Patents

Abstract

The invention discloses a new gene encoding the clonorchis sinensis class II 14-3-3 protein, a polypeptide encoded by the gene, and an antibody to the polypeptide. The invention also discloses that the polypeptide is used to screen the inhibitor of the Clonorchis sinensis class II 14-3-3 protein, and the application of the polynucleotide as a primer or as a probe, especially discloses that the polypeptide and the polynucleotide are used as diagnostic reagents box, vaccine use.

Description

Clonorchis sinensis class II 14-3-3 protein, its encoding nucleic acid and application thereof

technical field

The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide, the Clonorchis sinensis class II 14-3-3 protein, and the polynucleotide sequence encoding the polypeptide. The present invention also relates to the application of this polynucleotide and polypeptide.

Background technique

Clonorchis sinensis is a zoonotic disease. Adults parasitize in the liver and bile ducts. Adults will destroy the biliary epithelium and submucosal blood vessels, produce and secrete excretory antigens, cause inflammation in the bile duct and around the bile duct, and lead to local dilation of the bile duct and bile duct epithelial cells. hyperplasia. Some antigen components can also enter the liver tissue through the bile duct epithelial cells, causing inflammatory response and liver function damage. Long-term severe infection can lead to fibrous tissue hyperplasia and atrophic degeneration of liver cells, and even cause cancer, ascites and cirrhosis. Clonorchis sinensis occupies the bile duct, making bile outflow difficult, easy to be combined with bacterial infection and cholelithiasis.

The control of clonorchiasis mainly relies on comprehensive control measures. Strengthen the census and treatment of the population in the endemic areas, and strengthen the development of vaccines to reduce the source of infection and protect the susceptible population. At present, research on the prevention and control of Clonorchiasis sinensis focuses on diagnosis and vaccine candidate antigens, drug targets, and molecular mechanisms of pathogenicity.

14-3-3 proteins are small, acidic proteins of approximately 28-30 kDa ubiquitous in all eukaryotic cells. In 1968 Blake Moore and colleagues used starch electrophoresis in an attempt to identify specific proteins in the brain. The name 14-3-3 is due to the number of fragments and their location in the gel. In 1996, it was discovered that it binds to various proto-oncoproteins including Raf-1 and Bcr-AbL. It shows that 14-3-3 protein can bind various proteins related to signal transduction. These proteins include: Cdc25, NFAT, Bad, Cbl, A20, PI3-kinase, IRS-1, MEKK, p130Cas, adrenocorticoid receptor and some transcription factors. The list of 14-3-3-binding proteins is constantly growing, and these proteins have specific serine phosphorylation sequences, and most 14-3-3-binding proteins include such motifs.

14-3-3 proteins are involved in signal transduction, cell cycle regulation, vesicle trafficking and apoptosis, it is also involved in growth factor effects, DNA damage and tumorigenesis, for some enzymes 14-3-3 proteins act as a co-acting factors, which may act as inhibitors for other molecules. Recent studies have shown that 14-3-3 binding is involved in intracellular targeted trafficking. For nuclear proteins such as Cdc25 and NFAT, 14-3-3 binding keeps the protein in the cytoplasm and outside the nucleus, and for some apoptotic proteins such as BAD and A20, binding keeps them away from the cell membrane. For some proteins, 14-3-3 also has a chaperone-like effect. There are 2 isoforms in yeast, C. elegans and Drosophila, and 7 in mammalian cells, each of which seems to bind the same serine sequence, but 14-3-3 is expressed tissue-specifically, one of which One isoform σ can be regulated by p53 to block the cell cycle cycle. Due to the ubiquitous expression of 14-3-3 and the complex binding proteins, drug companies backed down, but the important role of the 14-3-3 protein family in cell cycle regulation and apoptosis indicated that 14-3-3 was partially blocked In combination with chemotherapeutic drugs that can potentially induce apoptosis.

(Andrey Shaw, The 14-3-3 proteins, Current Biology 10(11)400)

After extensive and in-depth research, the inventors isolated the DNA encoding the Clonorchis sinensis class II 14-3-3 protein from Clonorchis sinensis, expressed and purified the Clonorchis sinensis class II 14-3-3 protein, And further disclosed the application based on this polynucleotide and polypeptide.

Contents of the invention

One object of the present invention is to provide isolated novel polypeptides - Clonorchis sinensis class II 14-3-3 protein and fragments, analogs and derivatives thereof.

Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

Another object of the present invention is to provide an antibody against the polypeptide of the present invention—the Clonorchis sinensis class II 14-3-3 protein.

Another object of the present invention is to provide a polynucleotide that inhibits the expression of the polypeptide of the present invention.

Another object of the present invention is to provide the polypeptide of the present invention for screening inhibitors of Clonorchis sinensis class II 14-3-3 protein; or for identifying peptide fingerprints.

Another object of the present invention is to provide the application of the nucleic acid molecules of the present invention as primers for nucleic acid amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays.

Another object of the present invention is to provide a kit for detecting Clonorchis sinensis, which contains primers, antibodies or polypeptides for specifically detecting the Clonorchis sinensis class II 14-3-3 protein.

Another object of the present invention is to provide a vaccine for preventing Clonorchiasis sinensis, which contains the eukaryotic expression vector of the Clonorchis sinensis class II 14-3-3 protein or the polynucleotide encoding the protein.

In the first aspect of the present invention, the present invention relates to an isolated polypeptide, which is derived from Clonorchis sinensis, comprising: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant thereof, biological activity fragments or derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2. More preferably, the polypeptide does not contain a signal peptide sequence.

In a second aspect of the present invention, the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No.2;

(b) a polynucleotide complementary to polynucleotide (a);

More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having the sequence of positions 26-766 in SEQ ID NO: 1; and (b) having positions 1-1048 in SEQ ID NO: 1 the sequence of.

In the third aspect of the present invention, the present invention also relates to an antibody capable of specifically binding to the polypeptide of the present invention.

In a fourth aspect of the present invention, polynucleotides that inhibit the expression of the polypeptides of the present invention are provided.

In a fifth aspect of the present invention, uses of the polypeptides and coding sequences of the present invention are provided. For example, using polypeptides to screen inhibitors of Clonorchis sinensis class II 14-3-3 protein, the screened inhibitors are composed of safe and effective doses and pharmaceutically acceptable excipients for the treatment of Clonorchis sinensis A pharmaceutical composition; or for peptide fingerprint identification. The coding sequence or its fragments are used as primers for nucleic acid amplification reactions, or as probes for hybridization reactions, or for applications in the manufacture of gene chips or microarrays.

In the sixth aspect of the present invention, a kit for detecting Clonorchis sinensis is provided, which contains primers, antibodies or polypeptides for specifically detecting the Clonorchis sinensis class II 14-3-3 protein.

In the seventh aspect of the present invention, there is provided a vaccine for preventing Clonorchiasis sinensis, which contains the eukaryotic expression vector of Clonorchis sinensis class II 14-3-3 protein or polynucleotide encoding the protein.

Other aspects of the present invention will be apparent to those skilled in the art due to the disclosure of the technology herein. For example, the present invention may relate to a vector containing a polynucleotide of the present invention, particularly an expression vector; A vector genetically engineered host cell, including a transformed, transduced or transfected host cell; a method for producing a polypeptide of the invention comprising culturing said host cell and recovering an expression product.

Unless otherwise specified, the following terms used in this specification and claims have the following meanings:

"Nucleic acid sequence" means an oligonucleotide, nucleotide or polynucleotide, fragments or portions thereof, and may also refer to genomic or synthetic DNA or RNA, which may be single- or double-stranded, representing the sense strand or antisense strand. Similarly, the term "amino acid sequence" refers to oligopeptide, peptide, polypeptide or protein sequences and fragments or portions thereof. When "amino acid sequence" in the present invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with said protein molecule .

A protein or polynucleotide "variant" refers to an amino acid sequence or a polynucleotide sequence encoding it having one or more amino acid or nucleotide changes. The alterations may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. A variant may have "conservative" changes in which a substituted amino acid has similar structural or chemical properties to the original amino acid, eg, a leucine is replaced by an isoleucine. Variants may also have non-conservative changes, such as replacing glycine with tryptophan.

"Biologically active" refers to a protein that possesses the structural, regulatory, or biochemical functions of a native molecule. Similarly, the term "immunological activity" refers to the ability of a native, recombinant or synthetic protein and fragments thereof to induce a specific immune response and bind to specific antibodies in a suitable animal or cell.

"Inhibitor" refers to a molecule that blocks or modulates the biological activity of a Clonorchis sinensis class II 14-3-3 protein when bound to the Clonorchis sinensis class II 14-3-3 protein. Inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that bind to the Clonorchis sinensis class II 14-3-3 protein.

"Modulation" refers to a change in the function of the Clonorchis sinensis class II 14-3-3 protein, including an increase or decrease in protein activity, a change in binding properties, and any modification of the Clonorchis sinensis class II 14-3-3 protein. Alterations in other biological, functional or immune properties.

"Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the Clonorchis sinensis class II 14-3-3 protein using standard protein purification techniques. Substantially pure Clonorchis sinensis class II 14-3-3 protein yields a single major band on non-reducing polyacrylamide gels. The purity of the Clonorchis sinensis class II 14-3-3 protein polypeptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to the natural association of polynucleotides through base pairing under permissive conditions of salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or total. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

"Homology" refers to the degree of complementarity, which may be partial or complete. "Partially homologous"refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization under conditions of reduced stringency (Southern blot or Northern blot, etc.). Substantially homologous sequences or hybridization probes can compete and inhibit binding of a fully homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions of reduced stringency permit non-specific binding, since conditions of reduced stringency require the binding of two sequences to each other for a specific or selective interaction.

"Antisense" refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. "Antisense strand" refers to the nucleic acid strand that is complementary to the "sense strand."

"Derivative" refers to a chemical modification of HFP or the nucleic acid encoding it. Such chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain key biological properties of the native molecule.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fab, F(ab') ₂ and Fv, which can specifically bind to the epitope of the Clonorchis sinensis class II 14-3-3 protein.

The term "isolated" refers to the removal of a substance from its original environment (eg, its natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in a natural system is isolated. Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated Clonorchis sinensis class II 14-3-3 protein" means that the Clonorchis sinensis class II 14-3-3 protein is substantially free of other proteins, lipids, carbohydrates with which it is naturally associated or other substances. Those skilled in the art can purify the Clonorchis sinensis class II 14-3-3 protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the Clonorchis sinensis class II 14-3-3 protein polypeptide can be analyzed by amino acid sequence.

In the first aspect of the present invention, the present invention provides a new polypeptide-Clonorchis sinensis class II 14-3-3 protein, which is basically composed of the amino acid sequence shown in SEQ ID NO:2. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of the Clonorchis sinensis class II 14-3-3 protein. As used in the present invention, the terms "fragment", "derivative" and "analogue" refer to polypeptides that substantially maintain the same biological function or activity of the Clonorchis sinensis class II 14-3-3 protein of the present invention. Fragments, derivatives or analogs of polypeptides of the present invention may be: (I) one wherein one or more amino acid residues are substituted by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted which may or may not be encoded by the genetic code; or (II) one in which a certain group on one or more amino acid residues is replaced by another group comprising substituents; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused to the mature polypeptide ( Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the description herein such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence.

In a second aspect of the present invention, the present invention provides an isolated polynucleotide consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:2. The polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1. The polynucleotide of the present invention is discovered from a full-length cDNA library of Clonorchis adults. The full-length polynucleotide sequence it contains is 1050 bases, and its open reading frame (28-768) encodes 247 amino acids. According to the amino acid sequence homologous comparison, it is found that the polypeptide has 63% homology with the class II 14-3-3 protein of Caenorhabditis elegans, and it can be deduced that the protein is the class II 14-3-3 protein of Clonorchis sinensis.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used in the present invention, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein or polypeptide having SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 1.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: the coding sequence for the mature polypeptide only; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences. coding sequence.

The term "polynucleotide encoding a polypeptide" is intended to include polynucleotides encoding the polypeptide and polynucleotides including additional coding and/or non-coding sequences.

The present invention also relates to variants of the polynucleotides described above, which encode polypeptides having the same amino acid sequence as those of the present invention or fragments, analogs and derivatives of polypeptides. 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. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the sequences described above. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with Use a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, the length of "nucleic acid fragment" contains at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides glycosides or more. The nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding the Clonorchis sinensis class II 14-3-3 protein.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The specific polynucleotide sequence encoding the Clonorchis sinensis class II 14-3-3 protein of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, 2) antibody screening of expression libraries to detect cloned polynucleotides with common structural features Fragment, 3) polynucleotide fragment isolated by expressed sequence tag (EST) technology.

The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of the polypeptide.

Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The method of choice more often is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to generate a plasmid or phage full-length cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit is also available from commercial sources (Qiagene). And constructing a cDNA library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). When combined with polymerase reaction technology, even minimal expression products can be cloned.

These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) presence or loss of marker gene function; (3) measurement of transcription of Clonorchis sinensis class II 14-3-3 protein (4) Detect the protein product expressed by the gene by immunological techniques or measuring biological activity. The above methods can be used alone or in combination with multiple methods.

In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probes used here are usually DNA sequences chemically synthesized based on the gene sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the Clonorchis sinensis class II 14-3-3 protein gene. .

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain the full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein. selected and synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequences of the genes of the present invention obtained as described above, or various DNA fragments, etc., can be determined by conventional methods such as the dideoxy chain termination method. Such polynucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

The present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells produced by genetic engineering using the vectors of the present invention or directly using the Clonorchis sinensis class II 14-3-3 protein coding sequence, as well as those produced by recombinant techniques Methods for the polypeptides of the invention.

In the present invention, the polynucleotide sequence encoding the Clonorchis sinensis class II 14-3-3 protein can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria; pMSXND expression vectors expressed in mammalian cells and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational regulatory elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the Clonorchis sinensis class II 14-33 protein and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.

Those of ordinary skill in the art will know how to select appropriate vectors/transcriptional regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, the polynucleotide encoding the Clonorchis sinensis class II 14-3-3 protein or the recombinant vector containing the polynucleotide can be transformed or transfected into host cells to form a gene containing the polynucleotide or the recombinant vector Engineered host cells. The term "host cell" refers to 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 such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells, etc.

Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. An alternative is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant Clonorchis sinensis class II 14-3-3 protein. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding the Clonorchis sinensis class II 14-3-3 protein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide ;

(2). Cultivate host cells in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In step (2), the medium used in the culture can be selected from various conventional mediums according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

In step (3), the recombinant polypeptide can be encapsulated in the cell, expressed on the cell membrane, or secreted out of the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. These methods include but are not limited to: conventional refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic destruction, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In the third aspect of the present invention, the present invention provides methods for producing antibodies using polypeptides, fragments and derivatives thereof as antigens. These antibodies can be polyclonal or monoclonal. The present invention also provides an antibody against the antigenic determinant of the Clonorchis sinensis class II 14-3-3 protein. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

The production of polyclonal antibodies can be obtained by directly injecting the 14-3-3 protein of Clonorchis sinensis class II into immunized animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance the immune response, including but It is not limited to Freund's adjuvant and the like. Techniques for preparing monoclonal antibodies to Clonorchis sinensis class II 14-3-3 protein include, but are not limited to, hybridoma technology, triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, and the like. Chimeric antibodies combining human constant regions and nonhuman variable regions can be produced using existing techniques. The existing technology for producing single-chain antibodies can also be used to produce single-chain antibodies against Clonorchis sinensis class II 14-3-3 protein.

The antibody against the Clonorchis sinensis class II 14-3-3 protein can be used in immunohistochemical techniques to detect the distribution, secretion and excretion of the Clonorchis sinensis class II 14-3-3 protein in the biopsy specimen.

The monoclonal antibody binding to the Clonorchis sinensis class II 14-3-3 protein can also be labeled with a radioactive isotope, and injected into the body to track its location and distribution. The antibody in the invention can be used to prevent or block the pathological changes caused by the Clonorchis sinensis class II 14-3-3 protein.

In the fourth aspect of the present invention, oligonucleotides (including antisense RNA, antisense DNA, interfering RNA) and ribozymes that inhibit Clonorchis sinensis class II 14-3-3 protein mRNA are also within the scope of the present invention Inside. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds. Interfering RNA (iRNA) is a small double-stranded RNA molecule that can specifically degrade target gene mRNA in vivo to silence the gene. Its sequence is generally a complementary sequence after 70 bp downstream of mRNA. By simultaneously cloning the segment sequence and its reverse sequence into the same eukaryotic expression vector, and transfecting Clonorchis body or tissue cells, the RNA transcribed from the plasmid forms dsRNA, which can specifically degrade the iRNA of the target gene. The mRNA silencing technique can be used to treat abnormalities in cell proliferation, development, or metabolism due to non-expression or abnormal/inactive expression of Clonorchis sinensis glutathione S-transferase, thereby confirming the gene (physiological function of the protein).

In the fifth aspect of the present invention, the polypeptide of the present invention and its inhibitor can be directly used in the diagnosis and treatment of liver fluke disease.

Specifically, as far as the new Clonorchis sinensis class II 14-3-3 protein of the present invention is concerned, this protein is a regulatory protein with various functions in Clonorchis sinensis. After it enters the host body, it induces an immune response and can be used as a Potential target antigen for immunodiagnosis of clonorchiasis.

The present invention also provides a method for screening compounds to identify agents that inhibit the activity of Clonorchis sinensis class II 14-3-3 protein, which plays an important role in regulating various physiological activities of Clonorchis sinensis Its antagonist can effectively interfere with the whole life process of Clonorchis sinensis, leading to its dysfunction and death. Therefore, it can be used as an important drug target. The antagonists of the Clonorchis sinensis class II 14-3-3 protein include screened antibodies, compounds, receptor deletions and analogs. Antagonists of the Clonorchis sinensis class II 14-3-3 protein can bind to the Clonorchis sinensis class II 14-3-3 protein and eliminate its function, or inhibit the production of the class II 14-3-3 protein, or Combining with the active site of class II 14-3-3 protein makes the polypeptide unable to perform biological functions.

When screening compounds as inhibitors, the Clonorchis sinensis class II 14-3-3 protein can be added to a bioanalytical assay by measuring the effect of the compound on the Clonorchis sinensis class II 14-3-3 protein and its substrate. The effect of the interaction between them to determine whether the compound is an inhibitor or not. The polypeptide molecule capable of binding to the Clonorchis sinensis class II 14-3-3 protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the Clonorchis sinensis class II 14-3-3 protein molecule should generally be labeled.

The invention also relates to a diagnostic test method for quantitatively and locatingly detecting the level of the Clonorchis sinensis class II 14-3-3 protein. These assays are well known in the art and include immunological and immunohistochemical assays. The Clonorchis sinensis class II 14-3-3 protein level detected in the test can be used as an index of the infection degree of Clonorchis sinensis (worm negative).

The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cut physically, chemically or enzymatically, and then subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis, and better mass spectrometry analysis.

Polynucleotides encoding Clonorchis sinensis class II 14-3-3 proteins are also useful for various therapeutic purposes. The recombinant gene therapy vector can be designed to express the variant Clonorchis sinensis class II 14-3-3 protein to inhibit the activity of endogenous Clonorchis sinensis class II 14-3-3 protein. For example, a variant Clonorchis sinensis class II 14-3-3 protein can be a shortened Clonorchis sinensis class II 14-3-3 protein that lacks the catalytic center, although it can bind to downstream substrates, But lacks class II 14-3-3 protein activity. Or design a small antisense nucleic acid according to the polynucleotide sequence of the gene to interfere with the gene transcription process and post-transcription translation process, which can also play the role of Clonorchis sinensis protein metabolism and other physiological functions. The plasmid that inhibits the activity of endogenous recombinant class II 14-3-3 protein can enter the hepatic and bile ducts in an appropriate dosage form and manner, be swallowed by the clonorchis sinensis, and enter its syncytium to function.

The polynucleotide encoding the Clonorchis sinensis class II 14-3-3 protein can be used for the diagnosis of Clonorchis sinensis. The polynucleotide encoding the Clonorchis sinensis class II 14-3-3 protein can be used to detect the presence of the Clonorchis sinensis and the expression level of the class II 14-3-3 protein. For example, design primers according to the polynucleotide sequence, use polymerase chain reaction (PCR) to extract nucleic acid templates from the eggs of stool samples for gene amplification, and determine whether the size of the amplified polynucleotide fragment meets the predetermined value. Infection with Clonorchis sinensis. The technology is highly sensitive and specific, and can overcome the missed and false detections that are prone to occur in conventional morphological detection methods. The method of genetic detection can not only detect whether there is Clonorchis sinensis infection, but also can type Clonorchis sinensis and determine its subspecies or strains. The DNA sequence encoding the Clonorchis sinensis class II 14-3-3 protein can also be used for hybridization of biopsy samples to determine the expression status of the Clonorchis sinensis class II 14-3-3 protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with Clonorchis sinensis class II 14-3-3 protein-specific primers can also detect the transcripts of Clonorchis sinensis class II 14-3-3 protein.

The sequences of the invention are also valuable for chromosome identification. The sequence will specifically target a specific location of a Clonorchis testis chromosome and can hybridize to it. Currently, there is a need to identify the specific site of each gene on the chromosome. Currently, only a few chromosomal markers based on actual sequence data (repeat polymorphisms) are available to mark chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on chromosomes.

In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing each Clonorchis chromosome. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

The PCR mapping method of somatic heterozygous cells is a quick method to locate DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved using a set of fragments from a particular chromosome or a large number of genomic clones by a similar method. Other similar strategies that can be used for chromosome mapping include in situ hybridization, chromosome prescreening by flow sorting with markers, and hybridization preselection to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal mapping in a single step. Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data.

In the sixth aspect of the present invention, the diagnosis of clonorchiasis is mainly by using an immunological diagnostic kit in addition to routine pathogenic biological examination. There are two main types: enzyme-linked immunosorbent assay kit (ELISA) and rapid immune colloidal gold kit (ICT). The diagnosis of clonorchiasis can be done by detecting circulating antigens in serum or urine to determine current infection, or by detecting specific antibodies in serum or saliva for rapid screening. Since Clonorchis sinensis mainly parasitizes outside the tissue, most of the antigenic components produced are discharged into the intestinal tract along with the bile. Only when the degree of infection is high, the worm body seriously blocks the bile duct, and causes severe damage to the bile duct epithelium, it is secreted and excreted. Drugs can enter the liver and peripheral blood. Most of the circulating antigens entering the peripheral blood are neutralized by antibodies, and only a small amount of free circulating antigens can be detected. Therefore, methods to detect circulating antigens, although reflective of current infection, are less sensitive. At present, antibody detection kits are mainly used clinically for auxiliary diagnosis. There are two methods for detecting antibodies. One is to use labeled secondary antibody as a detection reagent to combine with the antibody captured by the antigen coated on the reaction plate or reaction membrane, and the other is to use the labeled antigen as a detection reagent. As a detection reagent, the secondary antibody can generally detect only one antibody, and has relatively high non-specific binding; as a detection reagent, the labeled antigen can detect multiple antibodies with higher specificity.

In the present invention, the purified Clonorchis sinensis class II 14-3-3 protein can be used to prepare an ELISA detection kit, and the purified Clonorchis sinensis class II 14-3-3 protein can also be used to prepare a colloidal gold immunochromatographic detection kit .

In the seventh aspect of the present invention, Clonorchis sinensis is an ectoparasite, and it is difficult for the immune system to directly interact with it. The antigen it produces stimulates the body's mucosal system to produce a mucosal immune response, which is its main protective response. The secretory IgA produced by mucosal immunity can prevent the secretion and excretion of antigens from entering the liver cells through the bile duct epithelium, thereby reducing the toxic effect of these antigenic components on the body, rather than directly attacking and killing the worms. Therefore, the vaccine of Clonorchis sinensis is mainly oral vaccine, the recombinant type II 14-3-3 protein protein is mixed with lecithin 1:10, added 10 times of normal saline solution, and ultrasonically oscillated to make liposomes , into slow-release capsules to make oral recombinant protein vaccines.

Description of drawings

Fig. 1 is the polyacrylamide gel electrophoresis pattern (SDS-PAGE) of the isolated Clonorchis sinensis class II 14-3-3 protein, 29kDa is the molecular weight of the protein, and the arrow points to the isolated protein band.

Figure 2 is a schematic diagram of colloidal gold immunochromatography

label

1 Test line 2 Quality control line 3 Glass fiber sheet

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1: Cloning of Clonorchis sinensis class II 14-3-3 protein

The total RNA of Clonorchis sinensis adults was extracted by one-step method of guanidine isothiocyanate/phenol/chloroform. Poly(A) mRNA was isolated from total RNA with Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. The cDNA fragment was directional inserted into the multiple cloning site of the pBSK (+) vector (product of Clontech) using the Smart cDNA cloning kit (purchased from Clontech), transformed into DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined with a Dye terminate cycle reaction sequencing kit (product of Perkin-Elmer) and an ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 2e05, was a new DNA. The insert cDNA fragment contained in this clone was determined bidirectionally by synthesizing a series of primers. The results show that the full-length cDNA contained in the 2e05 clone is 1050bp (as shown in SeqID NO: 1), and there is a 741bp open reading frame (ORF) from the 26p to 766bp, encoding a new protein (as shown in SeqID NO: 2 shown). We named this clone C002E05, and named the encoded protein as Clonorchis sinensis class II 14-3-3 protein.

Example 2: Homology retrieval of cDNA clones

With the sequence of the Clonorchis sinensis class II 14-3-3 protein of the present invention and the protein sequence encoded thereof, use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], homologous search in Genbank, Swissport and other databases. The gene with the highest homology to the polypeptide of the present invention is a known Caenorhabditis elegans class II 14-3-3 protein, and its accession number in Genbank is Y08715. The results showed that the identity was 63%; the similarity was 77%.

Example 3: Cloning of the gene encoding the Clonorchis sinensis class II 14-3-3 protein by RT-PCR

The total RNA of Clonorchis adultes was used as template, and oligo-dT was used as primer to carry out reverse transcription reaction to synthesize cDNA. After purification with Qiagene kit, PCR amplification was carried out with the following primers:

Primer1: 5'-CCCCCATCTGCCTTAGGCTACAAACAT-3' (SEQ ID NO: 3)

Primer2: 5'-TGTCATAACACAGTGTATTTCACAAAG-3' (SEQ ID NO: 4)

Primer1 is the forward sequence starting from the 1bp at the 5' end of SEQ ID NO:1;

Primer2 is the reverse sequence of the 3' end in SEQ ID NO:1.

Amplification reaction conditions: 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl ₂ , 200μmol/L dNTP, 10pmol primers, 1U Taq DNA polymer in a reaction volume of 50μl Enzyme (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer), the reaction was performed for 25 cycles under the following conditions: 94°C for 30 sec; 56°C for 30 sec; 72°C for 2 min. During RT-PCR, β-actin was set as positive control and template blank was set as negative control. The amplified product was purified with a kit from QIAGEN, and connected to a pCR vector (product of Invitrogen) with a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was completely identical to 1-1048bp shown in SEQ ID NO:1.

Example 4: Northern blot analysis of the expression of the Clonorchis sinensis class II 14-3-3 protein gene:

Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane. ³² P-labeled DNA probes were prepared by the random primer method using α- ³² P dATP. The DNA probe used is the PCR-amplified Clonorchis sinensis class II 14-3-3 protein coding region sequence (26bp to 766bp) shown in FIG. 1 . The 32P-labeled probe (about 2×10 ⁶ cpm/ml) was hybridized to the RNA-transferred nitrocellulose membrane overnight at 42°C in a solution containing 50% formamide-25mM KH ₂ PO ₄ ( pH7.4)-5*SSC-5*Denhardt's solution and 200 μg/ml salmon sperm DNA. After hybridization, the filters were washed in 1×SSC-0.1% SDS at 55° C. for 30 min. Then, analysis and quantification were performed with Phosphor Imager.

Example 5: In vitro expression, isolation and purification of recombinant Clonorchis sinensis class II 14-3-3 protein

According to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, design a pair of specific amplification primers, the sequence is as follows:

Primer3: 5'-AAA GGATCC ATGGTTTCCGACGAGTCGCGGGTTG-3' (Seq ID No: 5)

Primer4: 5'-AAA CTCGAG TCACTGGTCAGTTTCACGCTCCGAC-3' (Seq ID No: 6)

The 5' ends of these two primers contain BamHI and XhoI restriction sites respectively, followed by the coding sequences of the 5' and 3' ends of the target gene respectively, and the BamHI and XhoI restriction sites correspond to the expression vector plasmid pET-30a( +) Selective endonuclease site on (Novagen product, Cat.No.69865.3). The pBS-C002E05 plasmid containing the full-length target gene was used as a template for PCR reaction. The PCR reaction conditions were: 10 pg of pBS-C002E05 plasmid in a total volume of 50 μl, 10 pmol of primers Primer-3 and Primer-4 respectively, and 1 μl of Advantage polymerase Mix (product of Clontech Company). Cycle parameters: 94°C for 20s, 63°C for 30s, 68°C for 2min, a total of 25 cycles. The amplified product and plasmid pET-30a(+) were digested with XhoI and BamHI respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Escherichia coli DH5α by the calcium chloride method. After culturing overnight on an LB plate containing kanamycin (final concentration 30 μg/ml), positive clones were screened by colony PCR and sequenced. The positive clone (pET-C002E05) with the correct sequence was selected to transform the recombinant plasmid into Escherichia coli BL21(DE3)plySs (product of Novagen) by calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg/ml), the host strain BL21 (pET-C002E05) was cultured at 37°C to logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol/L, and the culture was continued for 5 Hour. Bacteria were collected by centrifugation, broken by ultrasonic waves, supernatant was collected by centrifugation, and chromatographically performed with an affinity chromatography column His.Bind Quick Cartridge (product of Novagen Company) capable of binding to 6 histidines (6His-Tag), to obtain Purified target protein Clonorchis sinensis class II 14-3-3 protein. After SDS-PAGE electrophoresis, a single band was obtained at 29kDa (Figure 1). Transfer the band to PVDF membrane and use Edams hydrolysis method to analyze the N-terminal amino acid sequence. As a result, the N-terminal 15 amino acids are completely identical to the N-terminal 15 amino acid residues shown in SEQ ID NO: 2.

The physicochemical property of embodiment 6 Clonorchis sinensis II class 14-3-3 protein

After the Clonorchis sinensis lactate dehydrogenase purified by affinity chromatography was excised with Throbinenterokinase enzyme, the carrier sequence was subjected to SDS-PAGE electrophoresis and the standard molecular weight curve, and the molecular weight was measured to be about 29kDa; the protein contained acidic amino acid residues There are 45 bases and 37 basic amino acid residues. Using the Amerham isoelectric focusing electrophoresis apparatus, the isoelectric point of the gel is measured to be 5.27 by the isoelectric focusing electrophoresis of pH3-10 and pH4-7 gradient fixed gels. The protein molecule is spherical in aqueous solution environment and is easily soluble in water. Its half-life is 30 hours in mammalian mitochondria, more than 20 hours in yeast cells, and more than 10 hours in E. coli. The structure of the protein is unstable.

Example 7 Production of Anti-Clonorchis sinensis Class II 14-3-3 Protein Antibody

Rabbits were immunized with 4 mg of recombinant Clonorchis sinensis class II 14-3-3 protein purified by affinity chromatography plus complete Freund's adjuvant, and the protein was boosted once 15 days later with incomplete Freund's adjuvant. The titer of antibody in rabbit serum was determined by ELISA on the titer plate coated with 15 μg/ml recombinant Clonorchis sinensis class II 14-3-3 protein. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a Sepharose4B column activated by cyanogen bromide, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation proved that the purified antibody could specifically bind to the 14-3-3 protein of Clonorchis sinensis class II. The antibody is a polyclonal antibody, and the titer with the recombinant antigen is above 1:32 in the agar immunological double expansion test, and the titer with the crude antigen of the parasite is above 1:16. The antibody can detect the circulating antigen of class II 14-3-3 protein in heavily infected Clonorchis sinensis patients.

Embodiment 8: The polynucleotide fragment of the present invention is used as the application of hybridization probe

Select suitable oligonucleotide fragments from the polynucleotides of the present invention to be used as hybridization probes in many ways, such as using the probes to hybridize with genome or cDNA libraries of normal tissues or pathological tissues from different sources to Identify whether it contains the polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence, and further use the probe to detect the presence of the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology Whether the expression in tissue cells is abnormal. From the polynucleotide SEQ ID NO of the present invention: 1, select oligonucleotide fragments as hybridization probes, the following principles and several aspects to be considered should be followed: the preferred range of probe size is 18-50 nucleotides ; GC content is 30%-70%, if it exceeds, non-specific hybridization will increase; there should be no complementary region inside the probe; those that meet the above conditions can be used as primary probes, and then further computer sequence analysis, including the primary probes Carry out homology comparison with its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complementary regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases If the bases are exactly the same, the primary probe should not be used generally; in addition, the replacement mutant sequence based on the designed probe fragment or its complementary fragment is used as the second probe.

Probe 1 (probe1), belonging to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (40Nt):

5'-GGAGATTATTATCGTTATTTGGCTGAAGTACAAACGGGAG-3' (Seq ID No: 7)

Probe 2 (probe2), belonging to the second type of probe, is equivalent to the substitution mutation sequence (40Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

5'-GGAGATTATTATCGTTATCTGGCTGAAGTACAAACGGGAG-3' (Seq ID No: 8)

Nucleic acid probes usually use filter hybridization methods, including dot blotting, Southern blotting, Northern blotting, and copying methods. They all use basically the same steps to hybridize after immobilizing the polynucleotide sample to be tested on the filter membrane. .

For other unlisted common reagents and their preparation methods related to the following specific experimental steps, please refer to the literature: DNAPROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experimental manuals such as "Molecular Cloning Experiments" Guide" (Second Edition in 1998) [US] Sam Brook et al., Science Press.

Steps: 1) Suspend and precipitate fresh or freshly thawed Clonorchis sinensis with cold homogenization buffer (0.25mol/L sucrose; 25mmol/L Tris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/LMgCl ₂ ) ( About 10ml/g), at 4°C, homogenize the tissue suspension with an electric homogenizer at full speed until the tissue is completely broken. The DNA in the tissue was extracted with phenol and precipitated with ethanol. When RNA contamination must be removed, add RNase A to the DNA solution with a final concentration of 100ug/ml, incubate at 37°C for 30 minutes to digest the RNA, and then re-extract the DNA. Preparation of sample membrane:

2) Take 4×2 nitrocellulose membranes (NC membranes) of appropriate size, and lightly mark the sample location and sample number on them with a pencil. Each probe needs two NC membranes for later experiments In the step, wash the membrane with high-intensity conditions and intensity conditions respectively. Take 15 microliters each of the control and the control, spot on the sample membrane, and dry at room temperature. Place on filter paper soaked with 0.1mol/L NaOH, 1.5mol/LNaCl for 5 minutes (twice), dry and place on filter paper soaked with 0.5mol/L Tris-HCl (pH7.0), 3mol/LNaCl for 5 minutes (twice) and let dry. Put it in clean filter paper, wrap it with aluminum foil, and dry it under vacuum at 60-80°C for 2 hours.

3) After the probe is labeled with ³² P by phosphokinase, it passes through a Sephadex G-50 column, and the amount of isotope is monitored by a liquid scintillation instrument, and combined

The collected solution of the first peak is ^{the 32} P-Probe to be prepared (the second peak is free γ- ³² P-dATP).

4) Pre-hybridization: Put the sample membrane in a plastic bag, add 3-10mg of pre-hybridization solution (10×Denhardts; 6×SSC, 0.1mg/ml CT DNA (calf thymus DNA).), seal the bag , shake in a water bath at 68°C for 2 hours.

5) Hybridization: Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a water bath at 42°C overnight.

6) Membrane washing:

High strength wash film:

Take out the hybridized sample membrane; wash in 2×SSC, 0.1% SDS, 40°C for 15 minutes (2 times); 0.1×SSC,

In 0.1% SDS, wash at 40°C for 15 minutes (twice); in 0.1×SSC, in 0.1% SDS, wash at 55°C for 30 minutes (twice), and dry at room temperature.

Low intensity wash:

Take out the hybridized sample membrane; wash in 2×SSC, 0.1% SDS, 37°C for 15 minutes (twice); 0.1×SSC, in 0.1% SDS, wash at 37°C for 15 minutes (twice); 0.1×SSC, In 0.1% SDS, wash at 40°C for 15 minutes (2 times),

Allow to dry at room temperature.

7) X-ray autoradiography:

-70°C, X-ray autoradiography (the pressing time depends on the radioactivity of the hybridization spots).

Experimental results:

In the hybridization experiment carried out by using low-intensity washing conditions, the radioactive intensity of the hybridization spots of the above four probes was not significantly different; while in the hybridization experiment carried out by using low-intensity washing conditions, the radioactive intensity of the hybridization spots of probe 1 was significantly stronger than that of The radioactive intensity of the other three probe hybridization spots. Probe 1 can thus be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.

Embodiment 9: Preparation of diagnostic kit

Preparation of ELISA detection kit: the purified Clonorchis sinensis class II 14-3-3 protein was made into 0.1mg/ml with carbonate coating buffer ((pH9.6), and each 96-well enzyme standard Add 50ul to the well, overnight at 4°C, after pouring off the coating solution, use 100ul of PBS-Tween20 solution containing 10% skimmed milk powder to block the redundant protein binding sites on the well wall, and pour off the blocking solution after blocking overnight at 4°C. Add 50ul of the serum to be tested into the reaction well of the plate, shake gently for 5 minutes, let it stand in a wet box at 37°C for 30-60 minutes, and wash with PBS-Tween20 washing solution for 3 times, 3 minutes each time; Oxidase or alkaline phosphatase-labeled goat anti-human IgG (1:1000 dilution 50ul), repeat the above binding and washing process, then add substrate and chromogenic reagent for color development, when the color development is obvious, add 2 % sulfuric acid solution to terminate the reaction. Visually observe or use a microplate reader to measure the reaction results.

Preparation of colloidal gold immunochromatography detection kit: Common immunochromatography systems include dipstick, card and cassette. The cassette immunochromatographic system has the same basic principle and structure as shown in Figure 2: it consists of a chromatographic system (the chromatographic membrane is connected to one or two absorbing layers) a solid-phase immune reaction system (the antigen or antibody is coated in a linear form) At a specific position on the chromatographic membrane as a test line or a quality control line), the sample and colloidal gold labeling reagent are added from one end, and under the capillary action of the membrane, they move along the membrane surface to the other end and are absorbed by the absorbing layer. The components to be tested in the sample and the colloidal gold detection reagent undergo an immunological binding reaction with the capture reagent bound to the membrane during the chromatography process, and stay on the coated line (including test line 1 and quality control line 2), showing A red reaction line. When the sample does not contain the component to be tested, there will be no color at the test line 1, while the quality control line 2 as a positive control will be red. In the dipping strip detection kit, add the lower end of the test strip to the sample solution to be tested and the labeled colloidal gold solution; in the card type and box type detection kits, the labeled colloidal gold is stored in a dry solid form on a glass fiber sheet at one end 3. For the card type, you only need to drop the solution to be tested and the buffer solution directly onto the glass fiber sheet, close the card, and the result can be judged by the observation window on the cover in a few minutes; for the box type, put the test strip into a small plastic box , the end of the colloidal gold corresponds to the circular sample hole, the sample and buffer solution are added from the sample hole, and the result is judged from the rectangular observation window after a few minutes.

In this detection kit, the chromatographic membrane is a mixed cellulose membrane with a pore size of 8 μm, and the purified Clonorchis sinensis class II 14-3-3 protein is coated on the far end of the absorbing layer as the detection line. The serum was coated on the proximal end of the absorbing layer as a quality control line, and the colloidal gold solution labeled with Clonorchis sinensis class II 14-3-3 protein was dropped onto the glass fiber sheet, dried, and placed on the other end of the chromatographic membrane.

Preparation and labeling of colloidal gold: make chloroaurate salt into a concentration of 0.01% with deionized ultrapure water, after boiling, quickly add 1% trisodium citrate solution (add 2ml per 100ml), keep it on fire quickly Shake and mix well, cook until the color of the solution turns wine red, remove from the fire, and make up the original volume with ultrapure water. The average particle size of the prepared colloidal gold is about 20nm. Adjust the pH of the prepared colloidal gold solution to 5.5 with 0.25mol/L potassium carbonate solution, add the purified class II 14-3-3 protein in an amount of 15μg/mL under stirring, and react for 10 minutes under a centrifugal force of 15000g After centrifugation for 30 minutes, the pellet was resuspended with 1/10 of the original volume of PBS (pH 7.4).

Example 10: Preparation of vaccine

Clonorchis sinensis is an extraorgan parasite, and it is difficult for the immune system to directly interact with it. The antigen it produces stimulates the body's mucosal system to produce a mucosal immune response, which is its main protective response. The secretory IgA produced by mucosal immunity can prevent the secretion and excretion of antigens from entering the liver cells through the bile duct epithelium, thereby reducing the toxic effect of these antigenic components on the body, rather than directly attacking and killing the worms. Therefore, the vaccine of Clonorchis sinensis is mainly oral vaccine. The recombinant type II 14-3-3 protein is mixed with lecithin at a ratio of 1:10, and 10 times of normal saline solution is added and ultrasonically oscillated to make liposomes. Pack into slow-release capsules to make oral recombinant protein vaccines.

Sequence Listing

<110> Sun Yat-Sen University

<120> Clonorchis sinensis class II 14-3-3 protein, its encoding nucleic acid and application thereof

<130>cs002e05

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<170>PatentIn version 3.1

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gctttgtgaa atacactgtg ttatgaca 1048

<210>2

<211>247

<212>PRT

<213>Clonorchis sinensis

<400>2

Met Val Ser Asp Glu Ser Arg Val Val Arg Glu Asn Ile Ser Asp Pro

1 5 10 15

Glu Ala Leu Leu Tyr Ile Ala Lys Leu Ala Glu His Ala Glu Arg Phe

20 25 30

Thr Asp Met Ala Ala Ala Met Lys Lys Phe Thr Glu Glu Leu Lys Lys Pro

35 40 45

Leu Ser Asn Asp Glu Arg Asn Leu Phe Ser Val Ala Tyr Lys Asn Val

50 55 60

Val Gly Ala Arg Arg Ser Val Trp Arg Val Ile Thr Ser Ile Lys Asn

65 70 75 80

Lys Asp Ser Glu Asp Glu Lys Ser Pro Thr Asn Glu Leu Arg Arg Lys

85 90 95

Ile Glu Asn Glu Leu Glu Gln Val Cys Lys Glu Val Leu Asn Ile Leu

100 105 110

Glu Lys Asn Ser Leu Pro Ser Glu Thr Thr Asp Asp Gly Leu Val Phe

115 120 125

Tyr His Lys Met Lys Gly Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Gln

130 135 140

Thr Gly Asp Lys Arg Asn Glu Ser Val Gln Lys Ser His Gln Ala Tyr

145 150 155 160

Glu Glu Ala Thr Glu Lys Ala Lys Gln Ser Leu Ser Glu Thr His Pro

165 170 175

Ile Arg Leu Gly Leu Ala Leu Asn Tyr Ser Val Phe Tyr Tyr Glu Ile

180 185 190

Glu Asn Asn Pro Asp Lys Ala Cys Glu Leu Ala Lys Ser Ala Phe Asp

195 200 205

Asn Ala Ile Ser Arg Leu Asp Gln Ile Lys Asp Glu Ser Tyr Lys Asp

210 215 220

Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr

225 230 235 240

Ser Glu Arg Glu Thr Asp Gln

245

<210>3

<211>27

<212>DNA

<213>Clonorchis sinensis

<400>3

cccccatctg ccttaggcta caaacat 27

<210>4

<211>27

<212>DNA

<213>Clonorchis sinensis

<400>4

tgtcataaca cagtgtattt cacaaag 27

<210>5

<211>34

<212>DNA

<213>Clonorchis sinensis

<400>5

aaaggatcca tggtttccga cgagtcgcgg gttg 34

<210>6

<211>34

<212>DNA

<213>Clonorchis sinensis

<400>6

aaactcgagt cactggtcag tttcacgctc cgac 34

<210>7

<211>40

<212>DNA

<213>Clonorchis sinensis

<400>7

ggagattatt atcgttattt ggctgaagta caaacggggag 40

<210>8

<211>40

<212>DNA

<213>Clonorchis sinensis

<400>8

ggagattatt atcgttatct ggctgaagta caaacggggag 40

Claims (9)

1. An isolated polypeptide - Clonorchis sinensis class II 14-3-3 protein, characterized in that it contains: a polypeptide with an amino acid sequence shown in SEQ ID NO: 2, or an active fragment or analog of a polypeptide thereof or derivatives. 2. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof; (b) A polynucleotide complementary to polynucleotide (a). 3. The polynucleotide according to claim 2, characterized in that the sequence of the polynucleotide comprises the sequence of 26-766 in SEQ ID NO: 1 or the sequence of 1-1048 in SEQ ID NO: 1 . 4. An antibody capable of binding to a polypeptide, characterized in that said antibody is capable of specifically binding to said Clonorchis sinensis class II 14-3-3 protein. 5. A class of polynucleotides that regulate the expression of polypeptides, characterized in that they are polynucleotides that inhibit the expression of the Clonorchis sinensis class II 14-3-3 protein, and have a multinucleated polynucleotide as shown in SEQ ID NO: 1 The antisense sequence of the nucleotide sequence or its fragment or the oligomeric double-stranded RNA consistent with its sequence. 6. The use of the polypeptide according to claim 1, characterized in that it is used for screening inhibitors of Clonorchis sinensis class II 14-3-3 protein; or for identifying peptide fingerprints. 7. The application of the polynucleotide according to any one of claims 2-3, characterized in that it is used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for making genes chip or microarray. 8. A kit for detecting Clonorchis sinensis, characterized in that it contains primers for specifically detecting the 14-3-3 protein of Clonorchis sinensis class II, the antibody described in claim 4, and the antibody described in claim 1. Any or a combination of polypeptides. 9. A vaccine for preventing clonorchiasis, which is characterized in that it contains the polypeptide according to claim 1 or the eukaryotic expression vector containing the polynucleotide according to claim 2 or 3.

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