CN1670194A - Calcineurin of clonorchis sinensis, its coding nucleic acid and application - Google Patents

Abstract

The invention relates to genes encoding Clonorchis sinensis Cobbold calcineurin, polypeptides encoded by the genes, antibody of the polypeptides. The invention also discloses the depressor of the polypeptides for screening clonorchis sinensis cobbold calcineurin, the use of polynucleotides as primer or as probe, in particular the use of the polypeptide and polynucleotide as diagnosis reagent kit, vaccine and pharmaceutical composition.

Description

The clonorchis sinensis calcineurin, its coding nucleic acid and application thereof

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---clonorchis sinensis calcineurin, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the application of these polynucleotide and polypeptide.

Technical background

Clonorchis sinensis is a zoonosis, and adult colonizes in the hepatic duct, and adult can be destroyed blood vessel under biliary tract epithelium and the mucous membrane, and produces secretion drainage antigen, causes the inflammatory reaction on every side of bile duct and bile duct, causes expansion of bile duct limitation and bile duct epithelial cell hyperplasia.Some antigenic components can also enter hepatic tissue by bile duct epithelial cell, and reaction and liver dysfunction cause inflammation.Secular severe infections can cause proliferation of fibrous tissue and hepatocellular atrophy sex change, even causes canceration, ascites and liver cirrhosis.Liver fluke occupies bile duct, and it is not smooth to make that bile flows out, and easily merges infectation of bacteria and cholelithiasis occurs.

The control of rot mainly relies on comprehensive preventive health measures.At the mass screening and mass treatment of popular district reinforcement to the crowd, and the development of reinforcement vaccine, to reduce contagium and protection Susceptible population.At present, the emphasis of rot study on prevention concentrates on diagnosis and vaccine candidate antigen, drug targets, morbific molecular mechanism research.

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---clonorchis sinensis calcineurin, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

Calcineurin is a kind of Ca in the eukaryotic cell ₂The serine/threonine protein Phosphoric acid esterase that +/CaM relies on, calmodulin and calcineurin form complex body, and calcium ion combines with calmodulin-calcineurin complex body, activates calcineurin.It is a heterodimer albumen, contains two subunits, is respectively to have the catalytic subunit A (CnA) of avtive spot and can regulate subunit B (CnB) with the Ca2+ bonded.The primary sequence of two subunits and dimeric quaternary structure from yeast to the Mammals high conservative.

Discover that calcineurin does not exist only in the lower eukaryotes such as yeast, and in multiple human causal organism, exist, as also separating in Leishmania, schistosomicide, worm, the plasmodium and detecting calcineurin.Calcineurin is distributed widely in the mammalian tissues, and as in the organ-tissues such as liver, kidney, marrow, spleen, lung, thymus gland, pancreas, Tiroidina distribution being arranged all, the level distribution in mammal brain is the highest.

Calcineurin participates in the signal transduction pathway of various kinds of cell reaction process and Ca2+ dependence, comprise immunne response, the cAMP second messenger's approach of being correlated with, apoptosis, the generation of myocardial hypertrophy and memory process etc., to get be its effect in the signal transduction of T cell activation the most widely in functional study to calcineurin.Calcineurin is the mediators of activated T nucleus factor transcription factor activatory key enzymes such as (NFAT) and T cell activation signal transduction.In the T cell, it regulates a series of cytokine expression, as κ 3,, TNF α,, NF κ B,, NF (P) and TGF β.In yeast, calcineurin can improve yeast to the tolerance of salt, Mn2+ and temperature and to the adjusting of Ca2+ pump, and participate in regulating sporulation, endochylema moves and process such as mating.

The activity of calcineurin can be suppressed by bad p0-357 A of immunosuppressive drug (CsA) and FK506, these two kinds of immunosuppressive drugs can combine with separately acceptor CyP and FKBP in the cell, calcineurin combines with CyP-CsA or FKBP-FK506 mixture, can suppress the dephosphorylation activity of calcineurin, therefore can cause the inhibition of T cell activation its substrate (as transcription factor NFAT).

Because the clonorchis sinensis calcineurin plays an important role in the body critical function as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify the clonorchis sinensis calcineurin albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new clonorchis sinensis calcineurin sample protein coding gene also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

The inventor has separated code clonorchis sinensis calcium mixing phosphatase DNA through extensive and deep research from clonorchis sinensis, and expression and purification the clonorchis sinensis calcineurin, and further disclosed application based on these polynucleotide and polypeptide.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide---the clonorchis sinensis calcineurin with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of this polypeptide of coding.

Another object of the present invention provides at polypeptide of the present invention---the antibody of clonorchis sinensis calcineurin.

Another object of the present invention has provided the polynucleotide that suppress expression of polypeptides of the present invention.

Another object of the present invention provides the inhibitor that polypeptide of the present invention is applied to screen the clonorchis sinensis calcineurin; Perhaps be used for the peptide finger print identification.

Another object of the present invention provides nucleic acid molecule of the present invention and is used for nucleic acid amplification reaction as primer, perhaps is used for hybridization as probe, perhaps is used to make the application of gene chip or microarray.

Another object of the present invention provides a kind of test kit that detects clonorchis sinensis, and it contains primer, antibody or the polypeptide of specific detection clonorchis sinensis calcineurin itself.

Another object of the present invention provides a kind of vaccine that is used to prevent clonorchiasis sinensis, and it contains the carrier for expression of eukaryon of the polynucleotide of clonorchis sinensis calcineurin or this enzyme of encoding.

Another object of the present invention provides a kind of pharmaceutical composition that is used for the treatment of clonorchiasis sinensis, and it contains the inhibitor of the clonorchis sinensis calcineurin that useful polypeptide of the present invention filters out.

In a first aspect of the present invention, the present invention relates to a kind of isolated polypeptide, this polypeptide is the clonorchis sinensis source, it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.More preferably, this polypeptide does not contain signal peptide sequence.

In a second aspect of the present invention, the invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;

(b) with polynucleotide (a) complementary polynucleotide;

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 108-683 position among the SEQ ID NO:1; (b) has the sequence of 1-887 position among the SEQ ID NO:1.

In a third aspect of the present invention, the invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

In a fourth aspect of the present invention, the polynucleotide that suppress expression of polypeptides of the present invention are provided.

In a fifth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.For example, utilize polypeptide to be applied to screen the inhibitor of clonorchis sinensis calcineurin, the inhibitor that is filtered out is formed the pharmaceutical composition of treatment clonorchiasis sinensis with safe and effective dosage and pharmaceutically acceptable excipient; Perhaps be used for the peptide finger print identification.Utilize encoding sequence or its fragment to be used for nucleic acid amplification reaction, perhaps be used for hybridization, perhaps be used to make the application of gene chip or microarray as probe as primer.

In a sixth aspect of the present invention, a kind of test kit that detects clonorchis sinensis is provided, it contains primer, antibody or the polypeptide of specific detection clonorchis sinensis calcineurin itself.

In a seventh aspect of the present invention, a kind of vaccine that is used to prevent clonorchiasis sinensis is provided, it contains the carrier for expression of eukaryon of the polynucleotide of clonorchis sinensis calcineurin or this enzyme of encoding.

In a eighth aspect of the present invention, a kind of pharmaceutical composition that is used for the treatment of clonorchiasis sinensis is provided, it contains the inhibitor of the clonorchis sinensis calcineurin that useful polypeptide of the present invention filters out.

Others of the present invention for example, utilize the present invention can relate to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector because disclosing of the technology of this paper is conspicuous to those skilled in the art; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The following term of using in this specification sheets and claims has following implication unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" inhibition " is meant when combining with the clonorchis sinensis calcineurin, a kind of molecule that seals or regulate the biologic activity of clonorchis sinensis calcineurin.Inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of clonorchis sinensis calcineurin.

" adjusting " is meant that the function of clonorchis sinensis calcineurin changes, and comprises the change of any other biological property, function or the immune property of the change of the rising of protein active or reduction, binding characteristic and clonorchis sinensis calcineurin.

" pure basically " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying clonorchis sinensis calcineurin of standard.Basically pure clonorchis sinensis calcineurin can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of clonorchis sinensis calcineurin polypeptide is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete suppress complete homologous sequence and target sequence the condition of severity degree reduction under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fab, F (ab ') ₂And Fv, its energy specificity is in conjunction with the antigenic determinant of clonorchis sinensis calcineurin.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating clonorchis sinensis calcineurin " is meant that the clonorchis sinensis calcineurin is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying clonorchis sinensis calcineurin of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of clonorchis sinensis calcineurin polypeptide can be used amino acid sequence analysis.

In a first aspect of the present invention, the invention provides a kind of new polypeptide---clonorchis sinensis calcineurin, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of clonorchis sinensis calcineurin.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of clonorchis sinensis calcineurin of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

In a second aspect of the present invention, the invention provides isolating polynucleotide, substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to prop up testis from China to be tied to form the full-length cDNA library of worm and to find.The polynucleotide sequence total length that it comprises is 887 bases, its open reading frame (108-683) 192 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and Ciona intestinalis calcineurin have 30% homology, it is exactly the clonorchis sinensis calcineurin that deducibility goes out this albumen.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to polynucleotide with sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ IDNO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of code clonorchis sinensis calcium mixing phosphatase.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

The special polynucleotide sequence of code clonorchis sinensis calcium mixing phosphatase of the present invention can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect the homologous polynucleotide sequence, 2) antibody screening of expression library is to detect the polynucleotide passage of the clone with common structure feature, 3) the isolating polynucleotide passage of expressed sequence tag (EST) technology.

Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage full-length cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of mensuration clonorchis sinensis calcineurin; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of clonorchis sinensis calcineurin genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

The method of using round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can be used ordinary method such as double deoxidating chain termination measuring.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly with the host cell of clonorchis sinensis calcineurin encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence of code clonorchis sinensis calcium mixing phosphatase can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on the T7 promotor of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna that contains code clonorchis sinensis calcium mixing phosphatase and the expression vector of suitable transcribing/translational control element.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance etc.

Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.

Among the present invention, the polynucleotide of code clonorchis sinensis calcium mixing phosphatase or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Alternative is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce the clonorchis sinensis calcineurin of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of code clonorchis sinensis calcium mixing phosphatase of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). in suitable medium, cultivate host cell;

(3). separation, protein purification from substratum or cell.

In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

In a third aspect of the present invention, the invention provides and use polypeptide, and fragment, derivative as antigen to produce the method for antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at clonorchis sinensis calcineurin antigenic determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The method of the available clonorchis sinensis calcineurin of the production of polyclonal antibody direct injection immune animal (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of the monoclonal antibody of preparation clonorchis sinensis calcineurin includes but not limited to hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.Can be with existing technology production with the variable region bonded chimeric antibody in human constant region and inhuman source.And the technology of existing manufacture order chain antibody also can be used for producing the single-chain antibody of anti-clonorchis sinensis calcineurin.

The antibody of anti-clonorchis sinensis calcineurin can be used in the immunohistochemistry technology, and distribution and the secretion of clonorchis sinensis calcineurin in polypide that detects in the biopsy specimen drained.

With the also available labelled with radioisotope of clonorchis sinensis calcineurin bonded monoclonal antibody, inject in the body and can follow the tracks of its position and distribution.Antibody among the present invention can be used for preventing or blocking the pathological change that the clonorchis sinensis calcineurin causes.

In a fourth aspect of the present invention, suppress the oligonucleotide (comprising sense-rna, antisense DNA, RNA interfering) of clonorchis sinensis calcineurin mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.RNA interfering (iRNA) thus be a class in vivo degrade specifically target gene mRNA make a bit of double stranded rna molecule of this gene silencing, its sequence generally is one section later complementary sequence of mRNA5 ' downstream 70bp.By this section sequence and reverse sequence thereof being cloned in the same carrier for expression of eukaryon simultaneously transfection China testis polypide or histocyte, the RNA formation dsRNA that this plasmid transcribes out, the iRNA of energy degrade specifically target gene.The mRNA silent technology can be used for treating because the nothing of clonorchis sinensis Triptide sulfurtransferase is expressed or cell proliferation, growth or the metabolic disturbance of unusual/non-activity due to expressing, thereby confirms gene (proteinic physiological function).

In a fifth aspect of the present invention, the inhibitor of polypeptide of the present invention and this polypeptide can be directly used in the diagnosis and the treatment of rot.

Specifically with regard to new clonorchis sinensis calcineurin of the present invention, it can see through epithelial duct and enter liver as the secretory protein of clonorchis sinensis, becomes the antigenic component of human body, by monitoring this albumen or its specific antibody, can diagnose clonorchiasis sinensis.The height of this antigenic content can reflect indirectly also whether human body infects the degree of this disease and infection.

The present invention also provides SCREENED COMPOUND to check the method for the active medicament of clonorchis sinensis calcineurin with evaluation, calcineurin participates in the signal transduction pathway of various kinds of cell reaction process and Ca2+ dependence in clonorchis sinensis, comprise immunne response, the cAMP second messenger's approach of being correlated with, apoptosis, its inhibitor can effectively disturb the clonorchis sinensis protein metabolism, causes its metabolism disorder and death, therefore, it can be used as an important drug targets.The antagonist of clonorchis sinensis calcineurin comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of clonorchis sinensis calcineurin can combine and eliminate its function with the clonorchis sinensis calcineurin, or suppressing calcineurin produces, or combines with the avtive spot of calcineurin and to make this polypeptide can not bring into play biological function.

In screening during as the compound of inhibitor, the clonorchis sinensis calcineurin can be added in the bioanalysis mensuration, interactional influence between clonorchis sinensis calcineurin and its substrate be determined whether compound is inhibitor by measuring compound.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with clonorchis sinensis calcineurin bonded peptide molecule obtains.During screening, generally tackle clonorchis sinensis calcineurin molecule and carry out mark.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization clonorchis sinensis calcineurin level.These tests are known in the art, comprise the mensuration and the immunologic assay of specific enzyme activity.The clonorchis sinensis calcineurin level that is detected in the test can be used as an index of clonorchiasis sinensis gradient of infection (worm negative).

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.

The polynucleotide of code clonorchis sinensis calcium mixing phosphatase also can be used for multiple therapeutic purpose.The gene therapy vector of reorganization can be designed for the clonorchis sinensis calcineurin of expressing variation, to suppress endogenic clonorchis sinensis calcineurin activity.For example, a kind of clonorchis sinensis calcineurin of variation can be the clonorchis sinensis calcineurin that shortens, lacked catalytic center, though can combine with the substrate in downstream, lacks the calcineurin activity.Perhaps according to one section little antisense nucleic acid of polynucleotide sequence design of this gene, the translation process after disturbing this gene transcription process and transcribing can play the effect of clonorchis sinensis protein metabolism and other physiological function equally.The active plasmid of this inhibition endogenous reorganization calcineurin can enter in the liver and gall road with suitable formulation and mode, is eaten by clonorchis sinensis, enters in its synplasm and works.

The polynucleotide of code clonorchis sinensis calcium mixing phosphatase can be used for the diagnosis of clonorchiasis sinensis.The polynucleotide of code clonorchis sinensis calcium mixing phosphatase can be used for detecting the existence of clonorchis sinensis and the expression level of calcineurin.As designing primer according to this polynucleotide sequence, from the worm's ovum of stool sample, extract the nucleic acid-templated gene amplification of carrying out with polymerase chain reaction (PCR), whether meet the infection that preset value is determined clonorchis sinensis according to the size of polynucleotide passage of amplification.This technology height sensitivity is special, can overcome incidental omission of conventional morphologic detection method and flase drop.The method of gene test not only can detect the infection that whether has clonorchis sinensis, and can also prop up the testis fluke to China and carry out somatotype, determines its subspecies or strain.The dna sequence dna of code clonorchis sinensis calcium mixing phosphatase also can be used for biopsy specimen is hybridized to judge the expression situation of clonorchis sinensis calcineurin.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect the clonorchis sinensis calcineurin with the special primer of clonorchis sinensis calcineurin.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar China testis karyomit(e) particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the PCR screening and contain each bar China chromosomal somatocyte hybrid cell of testis.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.

In a sixth aspect of the present invention, the diagnosis of clonorchiasis sinensis is except the Pathogen Biology inspection except routine, and main mode is to use the immunological diagnostic reagent box.Mainly contain two classes: enzyme linked immunological kit (ELISA) and tachysynthesis colloidal gold kit (ICT).The diagnosis of clonorchiasis sinensis can be judged existing disease infection by the circulating antigen that detects in serum or the urine, also can come rapid screening by the specific antibody that detects in serum or the saliva.Because clonorchis sinensis mainly is to colonize in outside the tissue, the antigenic component overwhelming majority that is produced has only the infectiosity height along with bile enters enteron aisle, and polypide is serious to bile duct obstruction, when causing the epithelial duct serious damage, its secretion movement just can enter in liver and the peripheral blood.The circulating antigen major part that enters in the peripheral blood is neutralized by antibody, has only the circulating antigen of small-amount free to be detected.Therefore, can reflect existing disease infection though detect the method for circulating antigen, susceptibility is lower.It is at present, clinical that what be used for auxiliary diagnosis mainly is antibody assay kit.The method that detects antibody is divided into two kinds, and the one, catch to such an extent that antibody combines as detection reagent with the antigen on being coated on Sptting plate or reaction film with mark two is anti-, a kind of is that the antigen of usefulness mark is as detection reagent.Two is anti-as detection reagent, generally can only detect a kind of antibody, and non-specific binding is than higher; Labelled antigen can detect multiple antibody as detection reagent, and specificity is higher.

The present invention can utilize the clonorchis sinensis calcineurin of purifying to prepare the ELISA detection kit, also can utilize the clonorchis sinensis calcineurin of purifying to prepare the colloidal gold immunochromatographiassay assay reagent box.

In a seventh aspect of the present invention, clonorchis sinensis is to organize epizoa, and immunity system is difficult to directly and its effect.It is its main aversion response that the antigenic stimulation body mucous membrane system that it produces produces mucosal immunoreaction.The secretory IgA that mucosal immunity produces can stop secretion to drain antigen and enter liver cell by epithelial duct, thereby reduce the toxic action of these antigenic components to body, rather than directly polypide be played the effect that kills and wounds of attacking.Therefore, the vaccine of clonorchis sinensis is based on the oral type vaccine, and the calcineurin albumen of will recombinating mixes with Yelkin TTS at 1: 10, adds 10 times normal saline solution, and liposome is made in ultrasonic concussion, and the slow releasing capsule of packing into is made the oral type recombinant protein vaccine.

In a eighth aspect of the present invention, the present invention also provides a kind of pharmaceutical composition for the treatment of clonorchiasis sinensis, it contains the inhibitor of the clonorchis sinensis calcineurin that useful polypeptide of the present invention filters out, and the inhibitor that is filtered out is formed the pharmaceutical composition of treatment clonorchiasis sinensis with safe and effective dosage and pharmaceutically acceptable excipient.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.If possible, this inhibitor is described.

The inhibitor medicaments composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Be applied to the patient amount and dosage range will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Description of drawings

Fig. 1 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating clonorchis sinensis calcineurin, and 22kDa is proteinic molecular weight, and the arrow indication is isolated protein band.

Fig. 2 is the colloidal gold immunochromatographimethod mode chart

Label

1 testing wire, 2 nature controlling lines, 3 glass fiber sheets

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of clonorchis sinensis calcineurin

Extract the total RNA of clonorchis sinensis adult with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure the sequence of all clones' 5 ' and 3 ' end with Dye terminate cycle reactionsequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 9H08 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 9H08 clone is 908bp (shown in SeqID NO:1), from 108bp to 686bp the open reading frame (ORF) of a 579bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after C009H08, encoded protein matter called after clonorchis sinensis calcineurin.

Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of clonorchis sinensis calcineurin of the present invention, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with peptides homologous of the present invention is a kind of known Ciona intestinalis calcineurin, number is BAB85848.1 in the access of Genbank.The result shows that both have certain homology, and its homogeny is 30%; Similarity is 48%.

Embodiment 3: with the gene of RT-PCR method clones coding clonorchis sinensis calcineurin

Propping up the total RNA of testis adult with China is template, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:

Primer1：5’-AGGATCTCAATGCGACAACGCGAA-3’ (SEQ?ID?NO：3)

Primer2：5’-AATATTATCACAATCATCAGCATTAAC-3’ (SEQ?ID?NO：4)

Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;

Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 54 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-887bp shown in the SEQ ID NO:1 are identical.

Embodiment 4:Northern blotting is analyzed clonorchis sinensis calcineurin expression of gene:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ gRNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA 2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is the clonorchis sinensis calcineurin coding region sequence (198bp to 686bp) of pcr amplification shown in Figure 1.Will ³²The probe of P mark (about 2 * 10 ⁶Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH ₂PO ₄(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.

Embodiment 5: vivoexpression, separation and the purifying of reorganization clonorchis sinensis calcineurin

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

Primer3：?5’-AAACTCGAGATGTGTGTGGAGATCACT-3’ (Seq?ID?No：5)

Primer4：?5’-CAGGGATCCTTAGCGATTTTTGGTGGA-3’ (Seq?ID?No：6)

5 ' end of these two sections primers contains EcoRI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, XhoI and BamHI restriction enzyme site are corresponding to expression vector plasmid pET-30a (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-C009H08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-C009H08 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 57 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-30a (+) are carried out double digestion with XhoI and BamHI, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-C009H08) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-C009H08) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein clonorchis sinensis calcineurin of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 1) at the 22kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.

The physicochemical property of embodiment 6 clonorchis sinensis calcineurins

With the liver fluke calcineurin of affinitive layer purification, after Throbin enzyme excision carrier sequence,, record its molecular weight and be about 22kDa through SDS-PAGE electrophoresis and standard molecule discharge curve; This albumen contains 28 acidic amino acid residues and 24 alkaline amino acid residues, utilizes Amerham isoelectric focusing electrophoresis instrument, and through the isoelectric focusing electrophoresis of pH3-10 and pH4-7 gradient fixing glue, recording its iso-electric point is 5.3.The transformation period of recombinant protein is 30 hours in the mammalian cell endoplasmic reticulum, surpasses 20 hours in yeast cell, surpasses 10 hours in intestinal bacteria, and protein molecular is spherical in aqueous environment, soluble in water, Stability Analysis of Structures.

Embodiment 7 anti-clonorchis sinensis calcineurin production of antibodies

Add the complete Freund's adjuvant immunizing rabbit with the clonorchis sinensis calcineurin, add the incomplete Freund's adjuvant booster immunization once with this albumen again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml reorganization liver fluke calcineurin bag quilt.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with calcineurin specifically.This antibody is polyclonal antibody, tires more than 1: 32 with recombinant antigen two the expansion in the test of agar immunity, tires more than 1: 16 with the reaction of polypide crude antigen.This antibody can detect the clonorchis sinensis patient's of severe infection calcineurin circulating antigen.

Embodiment 8: polynucleotide passage of the present invention is as the application of hybridization probe

Picking out suitable oligonucleotide fragment from polynucleotide of the present invention is of use in many ways as hybridization probe, as can whether containing polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence to identify it with the healthy tissues of different sources or the genome or the hybridization of cDNA library of pathological tissue with this probe, whether further also available this probe in detecting polynucleotide sequence of the present invention or the expression of its homologous polynucleotide sequence in healthy tissues or pathological tissue cell be unusual.Select oligonucleotide fragment as hybridization probe from polynucleotide SEQ ID NO:1 of the present invention, the several aspects that should follow following principle and need to consider: the probe size preferable range is a 18-50 Nucleotide; GC content is 30%-70%, and surpassing then, non-specific hybridization increases; Probe interior should not have complementary region; What meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use; In addition, again according to the probe fragment that designed or its complementary segmental replacement mutant nucleotide sequence as second probe.

Probe 1 (probe1) belongs to first kind probe, and complete homology of gene fragment or the complementation (40Nt) of SEQ ID NO:1:

5’-TTGATCCAGACGAGCCGGTGCGGGACACTATAAACTATTT-3’ (Seq?ID?No：7)

Probe 2 (probe2) belongs to the second class probe, is equivalent to gene fragment or its complementary segmental replacement mutant nucleotide sequence (40Nt) of SEQ ID NO:1:

5’-TTGATCCAGACGAGCAGGTGCGGGACACTATAAACTATTT-3’ (Seq?ID?No：8)

Nucleic acid probe adopts the filter hybridization method usually, comprises dot blotting, Southern blotting, Northern blotting and copy method etc., and they all are that polynucleotide sample to be measured is fixed on use essentially identical step crossover in back on the filter membrane.

Other unlisted common agents and the compound method thereof relevant with following concrete experimental procedure please refer to document: DNAPROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and molecular cloning laboratory manual books more commonly used are as works such as " molecular cloning experiment guide " (second edition in 1998) [U.S.] Sa nurse Brooker, Science Press.

Step: 1) with the fresh or fresh clonorchis sinensis that thaws cooled homogenate damping fluid (0.25mol/L sucrose; 25mmol/L Tris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/LMgCl ₂) precipitation that suspends (approximately 10ml/g), 4 ℃ with electric homogenizer homogenate tissue suspension at full speed, until tissue by broken fully.DNA with in the phenol extraction process tissue uses ethanol sedimentation then.In the time of must removing RNA and pollute, RNA enzyme A can be added in the dna solution, final concentration is 100ug/ml, 37 ℃ of insulations digestion in 30 minutes RNA, and then extracting DNA again.The preparation of sample film:

2) get 4 * 2 suitably nitrocellulose filters (NC film) of size, mark point sample position and sample number thereon gently with pencil, each probe needs two NC films, so that wash film with high strength condition and strength condition respectively in the experimental procedure of back.Draw and each 15 microlitre of contrast, put on the sample film, dry at room temperature.Placing to soak into has 0.1mol/LNaOH, and last 5 minute of filter paper (twice) of 1.5mol/LNaCl, drying to place to soak into has 0.5mol/L Tris-HCl (pH7.0), last 5 minute of filter paper (twice) of 3mol/LNaCl, dries.Be sandwiched in the clean filter paper, wrap, 60-80 ℃ of vacuum-drying 2 hours with aluminium foil.

3) probe phosphokinase mark ³²Cross Sephadex G-50 post behind the P, monitor isotopic weight, merge and be required preparation behind the collection liquid of first peak with liquid glimmer instrument ³²P-Probe (second peak for free γ- ³²P-dATP).

4) prehybridization: the sample film is placed plastics bag, adding 3-10mg prehybridization solution (10 * Denhardt ' s; 6 * SSC, 0.1mg/ml CT DNA (calf thymus DNA).), seal sack after, 68 ℃ of water-baths were shaken 2 hours.

5) hybridization: plastics bag is cut off one jiao, adds the probe prepare, seal sack after, 42 ℃ of water-baths are shaken and are spent the night.

6) wash film:

High strength is washed film:

Take out and hybridized good sample film; 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃; 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃; 0.1 * SSC among the 0.1%SDS, washes 30 minutes (2 times) for 55 ℃, room temperature is dried.

Low strength is washed film:

Take out and hybridized good sample film; 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃; 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃; 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃, room temperature is dried.

7) X-light autography:

-70 ℃, X-light autography (the compressing tablet time decides according to hybridization spot radioactivity is strong and weak).

Experimental result:

Adopt low strength to wash the hybrid experiment that the film condition is carried out, more than four strong and weak not obviously differences of probe hybridization spot radioactivity; And adopting low strength to wash the hybrid experiment that the film condition is carried out, the hybridization spot radioactive intensity of probe 1 obviously is better than the radioactive intensity of other three probe hybridization spots.Thereby available probe 1 is qualitative and analyze existence and the differential expression of polynucleotide of the present invention in different tissues quantitatively.

Embodiment 9: the preparation diagnostic kit

The preparation of ELISA detection kit: the clonorchis sinensis calcineurin of purifying is cushioned liquid with the carbonate bag, and ((pH9.6) is made into 0.1mg/ml, every hole as the 96 hole enzyme marks adds 50ul, 4 ℃ are spent the night, after outwelling coating buffer, with the unnecessary protein binding site of PBS-Tween20 solution 100ul sealing hole wall that contains 10% skim-milk, after spending the night, 4 ℃ of sealings outwell confining liquid.Add test serum 50ul in the enzyme plate reacting hole, jog is after 5 minutes, leaves standstill in 37 ℃ of wet boxes 30～60 minutes, shakes repeatedly with the PBS-Tween20 washings and washs each 3 minutes 3 times; Add the goat anti-human igg (1: 1000 diluent 50ul) of horseradish peroxidase or alkali phosphatase enzyme mark again, repeat above-mentioned combination and washing process, add substrate and chromogenic reagent then, when the appearance colour developing is obvious, add 2% sulphuric acid soln termination reaction.Visual inspection or with microplate reader assaying reaction result.

The preparation of colloidal gold immunochromatographiassay assay reagent box: there are three kinds of the bar of soaking formula (dipstick), card form and boxlikes in common immunochromatography system.Boxlike immunochromatography system has identical ultimate principle and structure shown in Figure 2: by chromatographic system (chromatographic film links to each other with one or two absorption layer) solid-phase immunity reactive system (antigen or antibody are wire and are coated on the chromatographic film certain location as testing wire or nature controlling line), sample and colloid gold label reagent add from an end, under the capillary action of film, move to the other end along the film surface, and be absorbed by the absorption layer.Immune association reaction takes place with the capture agent that is combined on the film in composition to be measured in the sample and gold-immunochromatographyreagent reagent for assay in the chromatography process, and rests on bag by on the line (comprising testing wire 1 and nature controlling line 2), demonstrates the response line of a redness.In sample, do not contain composition to be measured, then do not develop the color at testing wire 1 place, then apparent red as the nature controlling line 2 of positive control.Soaking bar formula detection kit successively adds the test strip lower end in the colloidal gold solution of analyte sample fluid and mark; In card form and the boxlike detection kit, the Radioactive colloidal gold of mark is with on the exsiccant solid form preservation glass fiber sheets 3 at one end, card form only needs solution to be measured and damping fluid directly are added drop-wise on the glass fiber sheets, the card that closes, viewing window judged result that can capping behind the several minutes; Boxlike is packed detector bar in the small plastic box, puts the corresponding circular well of an end of Radioactive colloidal gold, and sample and damping fluid add from well, behind the several minutes from rectangular viewing window judged result.

In this detection kit, chromatographic film is the cellulose mixture film of aperture 8 μ m, the clonorchis sinensis calcineurin of purifying is coated on the far-end of absorption layer as detection line, a China testis patient's positive serum is coated on the near-end of absorption layer as nature controlling line, the colloidal gold solution of mark clonorchis sinensis calcineurin is added drop-wise to the glass fiber sheets after drying, is placed on the other end of chromatographic film.

The preparation of Radioactive colloidal gold and mark: chlor(o)aurate is made into 0.01% concentration with deionized ultrapure water, after boiling, the citric acid three sodium solution of rapid adding 1%, (every 100ml adds 2ml), not from the rapid jolting mixing of fire, boil solution colour always and become the red recession fire of grape wine, supply original volume with ultrapure water.The about 20nm of median size of the Radioactive colloidal gold of preparation.The colloidal gold solution for preparing is transferred pH8.0 with the solution of potassium carbonate of 0.25mol/L, the calcineurin that under stirring state, adds purifying by the amount of 15 μ g/mL, reacted 10 minutes, centrifugal 30 minutes of the centrifugal force of 15000g, precipitation suspends again with the PBS (pH8.0) of original volume 1/10.

Embodiment 10: the preparation vaccine

Clonorchis sinensis is to organize epizoa, and immunity system is difficult to directly and its effect.It is its main aversion response that the antigenic stimulation body mucous membrane system that it produces produces mucosal immunoreaction.The secretory IgA that mucosal immunity produces can stop secretion to drain antigen and enter liver cell by epithelial duct, thereby reduce the toxic action of these antigenic components to body, rather than directly polypide be played the effect that kills and wounds of attacking.Therefore, the vaccine of clonorchis sinensis is based on the oral type vaccine, and the calcineurin albumen of will recombinating mixes with Yelkin TTS at 1: 10, adds 10 times normal saline solution, and liposome is made in ultrasonic concussion, and the slow releasing capsule of packing into is made the oral type recombinant protein vaccine.

Embodiment 11: pharmaceutical compositions

The activity of calcineurin can be suppressed by immunosuppressive drug, these two kinds of immunosuppressive drug Ciclosporin As (CsA) and FK506 can combine with separately acceptor CyP and FKBP in the cell, calcineurin combines with CyP-CsA or FKBP-FK506 mixture, can suppress the dephosphorylation activity of calcineurin, therefore can cause the inhibition of T cell activation its substrate (as transcription factor NFAT).This inhibitor can be water-soluble,, makes the oral type tablet or inhibitor is incapsulated as excipient with starch, glycerine etc., can be used for the treatment of clonorchiasis sinensis.

Sequence table

＜110〉Zhongshan University

＜120〉clonorchis sinensis calcineurin, its coding nucleic acid and application thereof

<130>cs009h08

<160>8

<170>PatentIn?version?3.1

<210>1

<211>887

<212>DNA

<213>Clonorchis?sinensis

<400>1

aggatctcaa?tgcgacaacg?cgaaatgcta?tattaaacct?catcaagcaa?gtagaacgac 60

aaaaaaagct?ccattttaac?aagacagagc?tgagtatttt?gctgcacatg?tgtgtggaga 120

tcactgggcg?tgaagtccgc?gacatgacgg?tggatgaatt?aaagacgttt?ctctacggaa 180

ccctcgacat?tacaaatccg?ttcagtctgg?atggactatg?ccgtgcttca?atgcagagca 240

ccaaacatag?cgcagccaag?aagacagtca?gtcccatcga?ttttgtgtgc?ctaatgtctg 300

tgctattacg?tggtactata?ggggaacgtg?ccgaggtggc?gttccgcatt?atggatcttg 360

attcggacgg?attgatcaga?cgaaacgtcg?aagttaggcg?gctgcttcat?gattcatttg 420

atgtgtcggt?ggcagcgcaa?aatccagaga?ttgatccaga?cgagccggtg?cgggacacta 480

taaactattt?gtgcgaaaag?ttgcagtgca?ccctcacgca?accaatctca?cttgaccgtt 540

tcaaaactct?ggcttacgag?gaaccctgga?tagtcgaatg?tttgcttcct?tgtatacctt 600

ccagagatga?ccaatctcgt?gtttcaaaca?cttatcagta?cacatgttcg?tttgccatct 660

ctcgaaaatc?caccaaaaat?cgctaaacag?tgaccaagct?gtattgcgag?tctctggaca 720

taccactccg?agaggcagta?accctcaata?cagaaccaat?ttgtcaaata?ttctcaggta 780

ctactttcac?ttaaattttt?cgacttattt?gggtttgcaa?aaatatgcat?caagcaccag 840

tgttctgcag?tgctattact?gttaatgctg?atgattgtga?taatatt 887

<210>2

<211>192

<212>PRT

<213>Clonorchis?sinensis

<400>2

Met?Cys?Val?Glu?Ile?Thr?Gly?Arg?Glu?Val?Arg?Asp?Met?Thr?Val?Asp

1 5 10 15

Glu?Leu?Lys?Thr?Phe?Leu?Tyr?Gly?Thr?Leu?Asp?Ile?Thr?Asn?Pro?Phe

20 25 30

Ser?Leu?Asp?Gly?Leu?Cys?Arg?Ala?Ser?Met?Gln?Ser?Thr?Lys?His?Ser

35 40 45

Ala?Ala?Lys?Lys?Thr?Val?Ser?Pro?Ile?Asp?Phe?Val?Cys?Leu?Met?Ser

50 55 60

Val?Leu?Leu?Arg?Gly?Thr?Ile?Gly?Glu?Arg?Ala?Glu?Val?Ala?Phe?Arg

65 70 75 80

Ile?Met?Asp?Leu?Asp?Ser?Asp?Gly?Leu?Ile?Arg?Arg?Asn?Val?Glu?Val

85 90 95

Arg?Arg?Leu?Leu?His?Asp?Ser?Phe?Asp?Val?Ser?Val?Ala?Ala?Gln?Asn

100 105 110

Pro?Glu?Ile?Asp?Pro?Asp?Glu?Pro?Val?Arg?Asp?Thr?Ile?Asn?Tyr?Leu

115 120 125

Cys?Glu?Lys?Leu?Gln?Cys?Thr?Leu?Thr?Gln?Pro?Ile?Ser?Leu?Asp?Arg

130 135 140

Phe?Lys?Thr?Leu?Ala?Tyr?Glu?Glu?Pro?Trp?Ile?Val?Glu?Cys?Leu?Leu

145 150 155 160

Pro?Cys?Ile?Pro?Ser?Arg?Asp?Asp?Gln?Ser?Arg?Val?Ser?Asn?Thr?Tyr

165 170 175

Gln?Tyr?Thr?Cys?Ser?Phe?Ala?Ile?Ser?Arg?Lys?Ser?Thr?Lys?Asn?Arg

180 185 190

<210>3

<211>24

<212>DNA

<213>Clonorchis?sinensis

<400>3

aggatctcaa?tgcgacaacg?cgaa 24

<210>4

<211>27

<212>DNA

<213>Clonorchis?sinensis

<400>4

aatattatca?caatcatcag?cattaac 27

<210>5

<211>27

<212>DNA

<213>Clonorchis?sinensis

<400>5

aaactcgaga?tgtgtgtgga?gatcact 27

<210>6

<211>27

<212>DNA

<213>Clonorchis?sinensis

<400>6

cagggatcct?tagcgatttt?tggtgga 27

<210>7

<211>40

<212>DNA

<213>Clonorchis?sinensis

<400>7

ttgatccaga?cgagccggtg?cgggacacta?taaactattt 40

<210>8

<211>40

<212>DNA

<213>Clonorchis?sinensis

<400>8

ttgatccaga?cgagcaggtg?cgggacacta?taaactattt 40

Claims (10)

1, a kind of isolated polypeptide-clonorchis sinensis calcineurin is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

2, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;

(b) with polynucleotide (a) complementary polynucleotide.

3, polynucleotide as claimed in claim 2, the sequence that it is characterized in that described polynucleotide include the sequence of 1-887 position among the sequence of 108-683 position among the SEQ ID NO:1 or the SEQ ID NO:1.

4, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with described clonorchis sinensis calcineurin specificity bonded antibody.

5, a class is regulated the polynucleotide of expression of polypeptides, it is characterized in that they are the polynucleotide that suppress the expression of described clonorchis sinensis calcineurin, and have the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences or the oligomerization double-stranded RNA consistent with its sequence.

6, the application of polypeptide according to claim 1 is characterized in that it is applied to screen the inhibitor of clonorchis sinensis calcineurin; Perhaps be used for the peptide finger print identification.

7, as the application of the described polynucleotide of arbitrary claim among the claim 2-3, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

8, a kind of test kit that detects clonorchis sinensis is characterized in that it contains the primer of specific detection clonorchis sinensis calcineurin, the described antibody of claim 4, the described polypeptide of claim 1 is arbitrary or its combination.

9, a kind of vaccine that is used to prevent clonorchiasis sinensis is characterized in that it contains the described polypeptide of claim 1 or contains claim 2 or the carrier for expression of eukaryon of 3 described polynucleotide.

10, a kind of pharmaceutical composition that is used for the treatment of clonorchiasis sinensis is characterized in that containing the inhibitor of the clonorchis sinensis calcineurin that the described polypeptide of useful claim 1 filters out.

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