CN101125884A - Polypeptide and its coding gene and application - Google Patents
Polypeptide and its coding gene and application Download PDFInfo
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- CN101125884A CN101125884A CNA2007100700973A CN200710070097A CN101125884A CN 101125884 A CN101125884 A CN 101125884A CN A2007100700973 A CNA2007100700973 A CN A2007100700973A CN 200710070097 A CN200710070097 A CN 200710070097A CN 101125884 A CN101125884 A CN 101125884A
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Abstract
The invention provides an opypeptide: tyrosine oxidase antigen epitope district gene-expression-associated proteins tyrC and coded gene and relevant application thereof. The opypeptide has an amino acid sequence which has a 95-100 percent of amino acid homology showed by SEQ NO: 1. The gene contains a nucleotide sequence which has a 70-100 percent of polynucleotide homology showed by SEQ NO:2. The beneficial effects of the invention lie in that: the efficient opypeptide which can appraise the autologous antibody in the serum of the vitligo patient- the tyrosine oxidase antigen epitope district gene-expression-associated proteins tyrC is provided and the application of the opypeptide for detecting the tyrosine oxidase antibody of pigmentary dermatosis, especially in the autologous antibody in the serum of vitligo patient is also provided. The effects set a foundation for the selection of novel medicine in the future.
Description
(1) technical field
The present invention relates to a peptide species: white tyrC of human tyrosinase epitope district gene expression associated protein and encoding gene thereof, and the application in the autoantibody in identifying patients with vitiligo serum of this peptide species.
(2) background technology
(tyrosinase is the key enzyme of melanocyte building-up process TYR) to human tyrosinase, is that a kind of molecular weight is the cuproprotein that contains of 75kD, and a part exists with the membrane bound enzyme form in the born of the same parents, and remaining exists with the film soluble form.Tyrosine associated protein 1 (TRP-1) and tyrosine associated protein 2 (TRP-2) and TYR structural similitude, the aminoacid sequence of its gene product is all 40% mutually, molecular weight much at one, tertiary structure has the highly conserved sequence cysteine residues, have the enzymic activity copper binding site, contain and stride the film district.TYR, TRP-1 and TRP-2 play crucial effects as autoantigen in leukodermic autoimmunization pathogenesis, can stimulate body to produce the unusual antityrosinase autoantibody that raises.
Vitiligo is the skin pigment disappearance property disease due to melanophore destroys, and patients with vitiligo spreads all over all over the world, accounts for 1～2% of world population.There are more than 1,000 ten thousand patients with vitiligo in China approximately as calculating with 1%, and the infringement that this disease is caused beauty treatment has had a strong impact on patient's Mental health and normal doings and employment and selected, and has reduced patient's quality of life, groups of people even commit suicide because of suffering from this disease.Because this disease pathogenesis is not clear and definite as yet, treatment is difficult at present.The morbidity that studies show that patients with vitiligo in a large number in recent years is very most of closely related with the autoimmune function disorder, and patients with vitiligo serum melanophore antibody titers and this disease reactivity and severity are closely related.Research thinks that patients with vitiligo serum melanophore antibody titers and this disease reactivity and severity are closely related, and 50% limitation patients with vitiligo serum has anti-melanocytic antibody, and general property vitiligo is then up to 93%, and the TYR autoantibody is particularly important.
The foreign scholar has adopted a lot of diverse ways to the research of vitiligo antibody, and especially to vitiligo self related antigen TYR, TRP-1 and TRP-2 have carried out big quantity research.Song etc.
[7]Use immunoblotting detection patients with vitiligo serum 61% antityrosinase antibody positive, Okamoto etc. are arranged
[8]Studies show that antityrosinase associated protein-2 has 67% positive rate in the patients with vitiligo serum, show that tyrosinase-related protein family is likely the important antigen relevant with the vitiligo state of an illness.
The domestic scholar of having adopts the immunohistochemical methods method that patients with vitiligo, non-patients with vitiligo and healthy human serum antagonism melanophore antibody are detected, and in the 20 routine patients with vitiligo of being checked, has 13 examples positive findings to occur, and positive rate is 65%.And there is the scholar that one section cDNA sequence of TRP-1 has been carried out gene clone, expresses and purifying, and utilize its expression product to prepare the polyclonal antibody in TRP-1B cell epitope district.
The B cell epitope that the method for exempting from has been identified human tyrosinase is put in application such as Kemp, they lay respectively at this proteic 240-255,289-294,295-300,435-447,461-479 amino acids place, wherein the homologous amino acid sequence in the 301-314 bit table position district of the 305-317 position of TYR-1 and TYR-2 is contained in 289-300 bit table position district.People's total length tyrosine oxidase is because molecular weight is bigger, and expressing difficulty increases, direct antigen expressed epitope peptide, and its molecular weight is little, and is simple in structure, removed non-epitope district, and higher specificity and susceptibility are arranged.
As mentioned above, human tyrosinase is the important antigen that produces autoantibody in the patients with vitiligo serum, thereby needs in this area to prepare always and a kind ofly more effectively can identify that the human tyrosinase epitope district gene expression associated protein of autoantibody in the patients with vitiligo serum is white.
(3) summary of the invention
The present invention is in order to provide a kind of pigmented dermatosis tyrosine oxidase antibody of effectively identifying especially to identify the polypeptide of autoantibody in the patients with vitiligo serum: white tyrC of human tyrosinase epitope district gene expression associated protein and encoding gene thereof, and the application in the autoantibody in identifying patients with vitiligo serum of this peptide species.
For reaching goal of the invention the technical solution used in the present invention be:
One peptide species: the white tyrC of human tyrosinase epitope district gene expression associated protein, containing with amino acid identity shown in the SEQNO:1 is 95～100% aminoacid sequence.
Because the singularity of aminoacid sequence; any fragment or its variant that contains the polypeptide of aminoacid sequence shown in the SEQNO:1; as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this polypeptide or polypeptide variants and aforementioned amino acid sequence homology all belong to the row of protection domain of the present invention more than 95%.Concrete, described change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of being replaced has structure similar to original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycine with tryptophane.The fragment of polypeptide of the present invention, derivative or analogue are meant and keep identical biological function or the active polypeptide of the white tyrC of human tyrosinase epitope of the present invention district's gene expression associated protein basically, can be under conditions: (I) one or more amino-acid residues be 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; (II) certain group on one or more amino-acid residues is replaced by other group; (III) mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; (IV) 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).
Preferably, described polypeptide is the aminoacid sequence shown in SEQNO:1.
Described polypeptide can be recombinant polypeptide, natural polypeptides or synthetic polypeptide, it can be the product of natural purifying, or the product of chemosynthesis, or use recombinant technology from protokaryon or eucaryon host (for example: bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated.Polypeptide of the present invention can also comprise or not comprise initial methionine residues.
The invention still further relates to a kind of gene of coding said polypeptide.It is 70～100% nucleotide sequence that described gene can contain with the homology of polynucleotide shown in the SEQNO:2.Preferably, described encoding gene is the nucleotide sequence shown in the SEQ NO:2.
Because the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQNO:2 as long as itself and this polynucleotide have 70% above homology, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide is meant a kind of polynucleotide sequence that one or more Nucleotide change that has.The variant of these polynucleotide can make the living allelic variant or the varient of non-life, comprises replacing varient, deletion mutation body and inserting varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of a plurality of Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
In addition; can (have 50% homology at least with the polynucleotide of the hybridization of polynucleotide sequence shown in the SEQNO:2; preferably have 70% homology), also at the row of protection domain of the present invention, particularly under stringent condition can with the polynucleotide of nucleotide sequence hybridization of the present invention.Described " 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% (VIV) methane amide, 0.1% calf serum, 0.1%Ficoll, 42 ℃; Or (3) only in the homology between the two sequences at least more than 95%, be more preferably more than 97% when being and just hybridize.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the polypeptide shown in the SEQ ID NO:1.
The special polynucleotide sequence of the white tyrC of human tyrosinase epitope district's gene expression associated protein of the present invention of encoding 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 with the antibody screening that detects homologous polynucleotide sequence and (2) expression library to detect the polynucleotide passage of the clone with common structure feature.Sequence dna fragment of the present invention also can obtain with following method: (1) separates double chain DNA sequence from genomic dna; (2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
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 the white tyrC of mensuration human tyrosinase epitope district's gene expression associated protein; (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.
The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can use the ordinary method dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.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, could splice the cDNA sequence of total length.
The invention still further relates to a kind of recombinant vectors that contains aforementioned gene, and the genetically engineered host cell that obtains with described recombinant vectors conversion, transduction or transfection.
Among the present invention, the polynucleotide sequence of the white tyrC of coding human tyrosinase epitope district's gene expression associated protein can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation." 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: and the expression vector based on the T7 promotor of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pcDNA3.1 carrier of in mammalian cell, expressing (Leeand Nathans, J Bio Chem.263:3521,1988) 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, preferred pGEX carrier series, pET carrier series and other prokaryotic expression carrier series.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 coding human tyrosinase epitope district's gene expression associated protein white tyrC and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambrook, et al., Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.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 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 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 that DNA expresses the cis acting factor, 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.
Among the present invention, the polynucleotide of coding human tyrosinase epitope district's gene expression associated protein white tyrC or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of this Nucleotide or recombinant vectors with formation." 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; Your bacterium of bacterial cell such as mouse typhus sramana; 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 the routine techniques that art technology is known 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 large intestine fourth bacterium, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used this area is well-known, and 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 white tyrC of human tyrosinase epitope district gene expression associated protein of reorganization.In general following steps are arranged:
(1), or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide with the polynucleotide (or varient) of the white tyrC of coding human tyrosinase epitope district's gene expression associated protein of the present invention;
(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 displacement 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.
Main points of the present invention have been to provide the nucleotide sequence shown in aminoacid sequence shown in the SEQ NO:1 and the SEQ NO:2, under the situation of known this aminoacid sequence and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and the acquisition of related vector, host cell, all be conspicuous to those skilled in the art.
Described polypeptide can be used for detecting pigmented dermatosis tyrosine oxidase antibody, is particularly useful for detecting autoantibody in the patients with vitiligo serum.The human tyrosinase autoantibody level that is detected, can with lay down a definition human tyrosinase in patients with vitiligo morbidity importance and be used to the autoimmune disorder of diagnosing human tyrosinase to work.
Concrete, described application method is as follows: with patients with vitiligo serum is sample, is contrast with healthy person serum, and by elisa plate, 4 ℃ are spent the night with the polypeptide bag behind the 1 μ g/mL purifying, and 1 * PBST washing pats dry; The patients serum's incubation that adds dilution, 1 * PBST flushing, add 1: 37 ℃ of incubation 30min of 5000HRP link coupled rabbit anti-human igg enzyme mark binding substances, 1 * PBST flushing, the colour developing of TMB lucifuge, termination reaction adopts 450nm and 630nm dual wavelength to measure light absorption value, adds 2 times of standard deviations with the mean value greater than healthy serum A value and is judged to the positive.
The polynucleotide of coding human tyrosinase epitope district's gene expression associated protein white tyrC can be used for the diagnosis with the relative disease of human tyrosinase.The unconventionality expression of the expression that the polynucleotide of coding human tyrosinase epitope district's gene expression associated protein white tyrC can be used for detecting human tyrosinase human tyrosinase whether or under morbid state.As the dna sequence dna of the white tyrC of human tyrosinase epitope district's gene expression associated protein that encodes can be used for biopsy specimen is hybridized to judge the expression of human tyrosinase.Hybridization technique comprises the Southern blotting, Nouthern 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 little array (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.
The sudden change that detects the white tyrC gene of human tyrosinase epitope district's gene expression associated protein also can be used for the disease of diagnosing human tyrosinase relevant.The form of the white tyrC of human tyrosinase epitope district gene expression associated protein sudden change comprises that the point mutation compared with the dna sequence dna of the white tyrC of normal wild type human tyrosinase epitope district's gene expression associated protein, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence protein expression, therefore uses the Nouthern blotting, and the Western blotting can judge indirectly that gene has or not sudden change.
Beneficial effect of the present invention is mainly reflected in: a kind of polypeptide that effectively can identify autoantibody in the patients with vitiligo serum is provided: human tyrosinase epitope district this polypeptide of gene expression associated protein bletilla is detecting the especially application in the autoantibody in the patients with vitiligo serum of pigmented dermatosis tyrosine oxidase antibody, for from now on new medicament screen provides the foundation.
(4) description of drawings
Fig. 1 is the plasmid map of recombinant plasmid pGEX-4T-2;
Fig. 2 is recombinant plasmid pGEX-tyrC restriction enzyme digestion and electrophoresis figure;
Fig. 3 is the SDS-polyacrylamide gel electrophoresis figure (SDS-PAGE) of the white tyrC of isolating human tyrosinase epitope district's gene expression associated protein.The arrow indication is isolated protein band and molecular weight thereof.
Fig. 4 is that the western trace of target protein detects.1: middle molecular weight protein dyes Marker in advance; The 2:BL21 expressed proteins; The GST albumen that 3:pGEX-4T-2 expresses; 4: the expressing protein of no IPTG inductive pGEX-tyrC; 5:IPTG induces the expression total protein of back pGEX-tyrC; The soluble protein that 6:IPTG induces back pGEX-tyrC to express; 7: the purpose purifying protein.
Fig. 5 is that patients with vitiligo serum IgG antibody titre detects.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
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 HarborLaboratory Press, 1989) condition described in, or according to the condition of the suggestion of manufacturer.Material and reagent:
PGEX-4T-2 plasmid and BL21 intestinal bacteria bacterial classification are so kind as to give by professor Zhang Chuanxi of insect science institute of Zhejiang University, and DH5 α intestinal bacteria bacterial classification is preserved by Molecular Biology Lab of Dermatology Department of No.3 People's Hospital, Hangzhou City..The TaqDNA polysaccharase is purchased in Shen, Shanghai energy lottery industry biotech firm; Restriction enzyme, T4 dna ligase, N,O-Diacetylmuramidase, isopropyl-(IPTG) and middle molecular weight protein matter marker are purchased in Shanghai bio-engineering corporation.Glutathione S-transferase (GST) mouse-anti monoclonal antibody is available from U.S. Upstate company, and the GST purification kit is purchased the Pierce company in the U.S..Coating buffer was a 0.2mol/L pH9.6 sodium carbonate salt buffering during ELISA detected; Confining liquid is phosphate buffered saline buffer (PBS) solution that contains 5% skim-milk, and washings is the PBS solution (PBST) that contains 0.5%Tween-20, and 3,3 ' 5,5-tetramethyl benzidine (TMB) solution are purchased the Pierce company in the U.S..
Embodiment 1: case is collected
From Dermatology Department of No.3 People's Hospital, Hangzhou City. outpatient service on March 1st, 2006 between 30 days September in 2006, do not merge other disease and patients with vitiligo serum 100 examples (general property 33 examples of obvious progress were arranged without immunosuppressant treatment, the state of an illness in nearly 3 months, limitation 24 examples, acra 17, being dispersed in property 26 examples), male 39 examples, women 61 examples, 17～57 years old age, 29 years old mean age, the course of disease 1 month to 10 years, average 3.1 years, normal human serum 30 examples, wherein male 13 examples, women 17 examples, 21～60 years old age, average 34 years old, all pick up from the health check-up healthy person.All are collected case and all obtain patient's informed consent signature and Ethics Committee's approval.
Embodiment 2: human tyrosinase epitope district gene tyrC's is synthetic
According to bibliographical information human tyrosinase epitope district 240-255,289-294,295-300,435-447,461-479, (the gene pool accession number: nucleotide sequence M27160) carries out synthetic, and introduces the BamHI restriction enzyme site at 5 ' end of gene according to the human tyrosinase full-length gene, introduce Xho I restriction enzyme site and termination codon subsequence at 3 ' end, synthetic gene tyrC size is 200bp.This work can lottery industry biotech firm be finished by the Shen, Shanghai.Gene after synthetic check order be listed as consistent with SEQID NO:2.
Embodiment 3: the structure of human tyrosinase epitope district gene tyrC expression vector
Amplification back tyrC fragment is purified, be connected to the BamHI/Xho I site (the recombinant plasmid collection of illustrative plates is seen Fig. 1) of prokaryotic expression carrier pGEX-4T-2 (being so kind as to give) behind BamHI/xhoI (purchasing) double digestion respectively by professor Zhang Chuanxi of insect science institute of Zhejiang University in Shanghai bio-engineering corporation, after T4 dna ligase (purchasing in Shanghai bio-engineering corporation) connects with in the recombinant plasmid transformed DH5 α competent escherichia coli cell, screening recombinant conversion, recombinant plasmid pGEX-tyrC after the screening is changed among the escherichia coli expression bacterial strain BL21 once more, and through PCR, enzyme is served the order-checking of Hai Shenggong company after cutting evaluation (restriction enzyme digestion and electrophoresis figure sees Fig. 2).The purpose fragment of inserting check order be listed as consistent with SEQ ID NO:2.
The abduction delivering and the purifying of embodiment 4:GST fusion rotein
When the bacterium liquid that will contain embodiment 3 gained recombinant plasmids shakes to A ≈ 0.4, add isopropyl-(IPTG, purchase in Shanghai bio-engineering corporation) to final concentration 1.0mmol/L (0.8～1.0mmol/L all can), 37 ℃ of inducing culture 4～8h collect thalline according to a conventional method.With the ultrasonic disruption cell, 4 ℃, 6s/ time * 3 times; 4 ℃ of centrifugal 5min of 12000rpm/min; Get cleer and peaceful precipitation respectively, add 4 * sample-loading buffer, carry out SDS-polyacrylamide gel electrophoresis (SDS-PAGE).Electrophoresis result shows, purpose fusion rotein size after the expression is 33KD, consistent with expection expression product size, its soluble protein accounts for 60% of purpose fusion rotein, the GST albumen size of expressing in the positive control is relative molecular mass 26000, and negative control does not have target protein to express (Fig. 2).
Gst fusion protein purification kit specification sheets operation according to U.S. Pierce company.The 250ml thalline suspends with the 10ml protein lysate, and centrifugal back supernatant adds the GST purification column, and the 500l lavation buffer solution is washed 3 times, uses 2ml elution buffer wash-out recombinant protein totally 4 times at last.Use gel analysis systems analysis purpose purifying protein, its purifying rate is 90% (Fig. 2).
Embodiment 5:Western trace testing goal Expression of Fusion Protein
Recombinant protein carries out being transferred on the nitrocellulose filter behind the 10%SDS-PAGE electrophoresis.Use mouse-anti GST monoclonal antibody (purchasing upstate company) (or the GST mono-clonal in other hosts sources or GST polyclonal antibody also can) in the U.S., hatch (1: 1000～1: 2000 all can) to be diluted to 1: 2000, two anti-horseradish peroxidase (HRP) the link coupled sheep anti-mouse antibodies (purchasing Santa Cruz Biotechnology company) (or the anti-antibody of the anti-GST of the HRP link coupled in other hosts sources one also can) that use in the U.S., hatch (1: 2000～1: 5000 all can) to be diluted to 1: 5000, the immune response band uses the super quick luminescent solution of ECL (purchasing the Applygen Technologies company in the U.S.) (or luminous substrate of other HRP) to detect, and experimental result is exposed to X line film.The immunoblotting result shows that purpose fusion rotein and soluble protein thereof all have a specific band at relative molecular mass 33000 places, and positive control has a specific band (Fig. 3) at relative molecular mass 26000 places.
Embodiment 6: the antigenicity in vitiligo detects and titer determination
Detect the human tyrosinase autoantibody in the 100 example progressive stage patients with vitiligo,, adopt indirect elisa method to detect this proteic antigenicity with the negative contrast of 30 routine healthy person serum.By elisa plate, is contrast with the tyr albumen (professor Ruth of Yale is so kind as to give) of prokaryotic expression with 1g/mL (0.5ug/mL～2.0ug/mL all can) embodiment 4 gained purifying protein bags, and 4 ℃ are spent the night, and 1 * PBST washing 3 times pats dry.Add 100 μ L patients serum incubation 1h, 1 * PBST flushing 3 times, add 37 ℃ of incubation 30min of 1: 5000 (1: 2000～1: 5000 all can) HRP link coupled rabbit anti-human igg (or other anti-human IgGs of originating) enzyme mark binding substances, 1 * PBST flushing 3 times, behind TMB (or optics chemical action substrate of other HRP) the lucifuge colour developing 30min, 2mol/L H
2SO
4Termination reaction adopts 450nm and 630nm dual wavelength to measure light absorption value.With the mean value greater than healthy serum A value add 2 times of standard deviations (± 2S) be judged to the positive.Mean relatively adopts the independent sample t check between group, gets P=0.05 or 0.01 and is inspection level.
The result shows, in the progressive stages 100 routine patients with vitiligo, 64 routine serum all can have association reaction with target protein tyrC and tyr, positive rate is 64.0%, there is not positive reaction in the 30 routine normal healthy controls serum, statistical analysis shows that the positive detection of tyrC and tyr goes out there was no significant difference between the rate (P＞0.05).
Embodiment 7:
Choose the antibody titers experiment that 10 routine positive patient serum and healthy serum carry out dilution in 1: 10 to 1: 1280 respectively.Every part of serum to be checked is done independent repeated experiments 3 times.The gained data are represented with the mean value of A value, use the SPSS13.0 statistical package and carry out statistical analysis.Mean relatively adopts the independent sample t check between group, gets P=0.05 or 0.01 and is inspection level.
Positive patient that two kinds of target protein tyrC of comparison and tyr detect and healthy patients IgG antibody are in the A value of dilution in 1: 10 to 1: 1280, the result shows, when extension rate during at 1: 10 to 1: 640, the A value has significant difference (P＜0.05) between two groups of samples, when extension rate reached 1: 1280, there was no significant difference between two groups of samples (P＞0.05) (Fig. 4).Statistical analysis shows, there was no significant difference between the antibody titers that the positive patient of tyrC and tyr detects (P＞0.05).
Conclusion:
TyrC of the present invention compares with existing tyr and has the following advantages: the purpose fusion protein molecule amount of tyrC little (about 33kDa), expression condition is easier to grope, under optimal conditions, be difficult for forming inclusion body, majority is a soluble protein, therefore more easily obtain large-scale purification albumen, the purifying antigen of capacity can be provided for the application of test kit, and cost is also lower; And the purpose fusion protein molecule amount of tyr big (about 100kDa), expression condition is difficult for groping, even under optimal conditions, also be difficult for obtaining soluble protein, and the therefore difficult large-scale purification albumen that obtains, if be applied to the exploitation of test kit, under equal conditions can raise the cost greatly.
SEQUENCE?LISTING
＜110〉Xu Aie closes Cuiping, No.3 People's Hospital, Hangzhou City.
＜120〉peptide species and encoding gene thereof and application
<130>
<160>2
<170>PatentIn?version?3.4
<210>1
<211>61
<212>PRT
<213>Unknown
<220>
＜223〉synthetic
<400>1
Gly?Ser?Ala?Ala?Asp?Ala?Glu?Lys?Cys?Asp?Ile?Cys?Thr?Asp?Glu?Tyr
1 5 10 15
Met?Gly?Gly?Gln?Cys?Asn?Gly?Thr?Pro?Glu?Gly?Pro?Leu?Arg?Arg?Asn
20 25 30
Asn?Gly?Asp?Phe?Phe?Ile?Ser?Ser?Lys?Asp?Leu?Gly?Tyr?Gln?Asp?Tyr
35 40 45
Ile?Lys?Ser?Tyr?Leu?Glu?Gln?Ala?Ser?Arg?Ala?Leu?Glu
50 55 60
<210>2
<211>183
<212>DNA
<213>Unknown
<220>
＜223〉synthetic
<400>2
ggatccgccg?ccgatgcaga?aaaatgtgac?atttgcaccg?atgagtacat?gggtggtcag 60
tgcaatggta?cgccggaagg?tcctttacgt?cgtaataatg?gtgatttctt?tatttcatcc 120
aaagatctgg?gctatcagga?ctacattaaa?tcctatttgg?aacaagcgag?tcgtgcgctc 180
gag 183
Claims (10)
1. a peptide species: the white tyrC of human tyrosinase epitope district gene expression associated protein, containing with amino acid identity shown in the SEQ NO:1 is 95～100% aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that described polypeptide is the aminoacid sequence shown in SEQ NO:1.
3. gene of polypeptide according to claim 1 of encoding.
4. gene as claimed in claim 3 is characterized in that it is 70～100% nucleotide sequence that described gene contains with polynucleotide homology shown in the SEQ NO:2.
5. gene as claimed in claim 3 is characterized in that described gene is the nucleotide sequence shown in the SEQ NO:2.
6. recombinant vectors that contains just like claim 3 or 4 described genes.
7. genetically engineered host cell that obtains with recombinant vectors conversion as claimed in claim 6, transduction or transfection.
8. the application of polypeptide as claimed in claim 1 or 2 in detecting pigmented dermatosis tyrosine oxidase antibody.
9. application as claimed in claim 8 is characterized in that: described polypeptide is the application in the autoantibody in detecting patients with vitiligo serum.
10. application as claimed in claim 9 is characterized in that described application method is as follows: with patients with vitiligo serum is sample, is contrast with healthy person serum, and by elisa plate, 4 ℃ are spent the night with the polypeptide bag behind the 1 μ g/mL purifying, and 1 * PBST washing pats dry; The patients serum's incubation that adds dilution, 1 * PBST flushing, add 1: 37 ℃ of incubation 30min of 5000HRP link coupled rabbit anti-human igg enzyme mark binding substances, 1 * PBST flushing, the colour developing of TMB lucifuge, termination reaction adopts 450nm and 630nm dual wavelength to measure light absorption value, adds 2 times of standard deviations with the mean value greater than healthy serum A value and is judged to the positive.
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| CNA2007100700973A CN101125884A (en) | 2007-07-19 | 2007-07-19 | Polypeptide and its coding gene and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100700973A CN101125884A (en) | 2007-07-19 | 2007-07-19 | Polypeptide and its coding gene and application |
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| CN101125884A true CN101125884A (en) | 2008-02-20 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560919A (en) * | 2015-01-21 | 2015-04-29 | 华中农业大学 | Saccharomycopsis fibuligera polyclonal antibody and preparation method thereof |
| CN110226946A (en) * | 2019-06-12 | 2019-09-13 | 杭州市第三人民医院 | Urological surgery specimen trap |
-
2007
- 2007-07-19 CN CNA2007100700973A patent/CN101125884A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560919A (en) * | 2015-01-21 | 2015-04-29 | 华中农业大学 | Saccharomycopsis fibuligera polyclonal antibody and preparation method thereof |
| CN104560919B (en) * | 2015-01-21 | 2017-10-10 | 华中农业大学 | A kind of saccharomycopsis fibuligera bacterium polyclonal antibody and preparation method |
| CN110226946A (en) * | 2019-06-12 | 2019-09-13 | 杭州市第三人民医院 | Urological surgery specimen trap |
| CN110226946B (en) * | 2019-06-12 | 2022-05-17 | 杭州市第三人民医院 | Urinary surgery operation specimen collector |
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