CN101955528B - Human MCHR1 epitope domain gene expression-associated protein MCHR1-C and encoding gene thereof - Google Patents
Human MCHR1 epitope domain gene expression-associated protein MCHR1-C and encoding gene thereof Download PDFInfo
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Abstract
The invention provides a human melanin concentrating hormone receptor-1 (MCHR1) epitope domain gene expression-associated protein MCHR1-C, an encoding gene and a recombinant vector thereof and an application of the polypeptide in identifying antoantibody in serum of a vitiligo patient. The human MCHR1 epitope domain gene expression-associated protein MCHR1-C of the invention has an amino acid sequence shown in SEQ ID No:1. The human MCHR1 epitope domain gene expression-associated protein MCHR1-C of the invention has the beneficial effects that an polypeptide capable of effectively identifying the antoantibody in the serum of the vitiligo patient is provided; and the application of the human MCHR1 epitope domain gene expression-associated protein and the polypeptide in detecting MCHR1 antibody of pigmentary dermatosis, in particular the autoantibody in the serum of the vitiligo patient, thereby laying foundation for further new drug screening.
Description
(1) technical field
The present invention relates to a kind of people's melanin concentrating hormone receptor-1 (Melanin Concentrating Hormone Receptor 1; MCHR1) white MCHR1-C of epitope district gene expression associated protein and encoding sox thereof, recombinant vectors, and the application in the autoantibody in identifying patients with vitiligo serum of this peptide species.
(2) background technology
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 selected with normal doings and employment, has reduced patient's quality of life, groups of people even should disease commit suicide because of suffering from.Because this disease pathogenesis is not clear and definite as yet, treatment is difficult at present.Big quantity research in recent years shows that the morbidity of patients with vitiligo is very most of closely related with the autoimmune function disorder.Anti-melanocytic autoantibody is arranged in the patients with vitiligo serum, and anti-melanophore antibody titers is closely related with this disease reactivity and severity, 50% limitation patients with vitiligo serum has the melanocytic antibody of resisting, and general property vitiligo is then up to 93%.
The melanophore autoantigen comprises endochylema antigen and membrane antigens, and clear and definite endochylema antigen is TYR GAP-associated protein GAP family at present.TYR is that a kind of relative molecular mass is 75000 the cuproprotein that contains, and research shows that TYR GAP-associated protein GAP family is likely the important antigen relevant with the vitiligo state of an illness.The B cell epitope of the people TYR that basis has been reported in early-stage Study of seminar; The encoding sox that has synthesized 5 epi-position districts; Through having obtained relative molecular mass behind prokaryotic expression, the purifying is 33000 purpose fusion rotein TYR-C, and this purpose fusion rotein has the ability that combines the patients with vitiligo serum IgG.A large amount of identification research to membrane antigens show that MCHR1 is present unique clear and definite membrane antigens.Melanin concentrating hormone (MCH) is a kind of 19 amino acid whose neuropeptides that contain; Mainly be present in hypothalamus; It has two kinds of melanin concentrating hormone receptor MCHR1 and MCHR2, and the distribution of these two kinds of acceptors and the expression in brain are similar basically, but MCHR2 is not present in the rodent.MCHR1 belongs to g protein coupled receptor family, and (α-MSH) can cause that melanocyte is synthetic after combining with α-melanocyte yield stimulant for it.MCH is the antagonist of α-MSH, and can suppress melanic synthetic after acceptor MCHR1 combines, so the MCH/MCHR1 signal pathway plays an important role in synthetic regulating the melanophore melanocyte.The vitiligo autoantibody needn't can cause the negative effect to function of receptors through cytolemma, finally causes the destruction of eumelanin cell.
Kemp equals 2002, and (J Clin Invest, 2002,109 (7): 923-930) discovery MCHR1 is the relevant melanophore antigen of a new vitiligo, and vitiligo is had high degree of specificity, causes many scholars' concern.He Qiuhao equals 2005 (Chinese skin cypridology magazine; 2005,19 (2): 77-79 changes 88) made up the MCHR1 carrier for expression of eukaryon, and obtained the eukaryotic expression albumen of MCHR1; With this albumen is antigen, and anti-MCHR1 antibody positive rate is 17.1% in the detection patients with vitiligo serum; The same year; (Chinese journal of dermatology such as He Qiuhao; 2005,38 (1): 29-31) also expressed the B cell epitope peptide section of 1-94 amino acids among the MCHR1, obtained prokaryotic expression recombinant protein GST-MCHR1; With it is antigen, and 13.2% patients with vitiligo serum anti MCHR1 autoantibody IgG is positive.Kemp equals 2009 (Experimental Dermatology.2009; 18 (5): the B cell antigen epi-position district that 454-463) has identified MCHR1 is positioned at 51-80 respectively; 85-98; 154-158 and 254-260 amino acids, wherein major antigen epi-position district is positioned at 51-80 and 254-260 amino acids, and minor antigen epi-position district is positioned at 85-98 and 154-158 district.
The present invention is an antigen with the GST-MCHR1 purifying protein of 4 B cell epitopes containing MCHR1; Anti-MCHR1 autoantibody positive rate is 36% in the detection patients with vitiligo serum; The anti-MCHR1 autoantibody that is higher than existing research detects positive rate; This maybe all to come from progressive stage relevant with selected patients serum to be checked, also maybe be relevant with 4 epi-positions that the purpose purifying protein comprises.People's total length MCHR1 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 stated; People MCHR1 is the main membrane antigens that produces autoantibody in the patients with vitiligo serum, thereby need prepare in this area always and a kind ofly more effectively can identify that the people MCHR1 epitope district gene expression associated protein of anti-membrane antigens autoantibody in the patients with vitiligo serum is white.
(3) summary of the invention
The object of the invention is in order to provide a kind of pigmented dermatosis MCHR1 antibody of effectively identifying especially to identify the polypeptide of autoantibody in the patients with vitiligo serum: people MCHR1 epitope white MCHR1-C of district's gene expression associated protein and encoding sox thereof, and the application in the autoantibody in identifying patients with vitiligo serum of this peptide species.
The technical scheme that the present invention adopts is:
The white MCHR1-C of a kind of people MCHR1 epitope district's gene expression associated protein has the aminoacid sequence shown in the SEQID NO:1.
Because the singularity of aminoacid sequence; Any fragment or its variant that contains the polypeptide of aminoacid sequence shown in the SEQ NO:1; Like its examples of conservative variations, bioactive fragment or verivate; 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, said 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 with original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycocoll with tryptophane.The fragment of polypeptide according to the invention, verivate or analogue are meant and keep identical biological function or the active polypeptide of the white MCHR1-C of people MCHR1 epitope district's gene expression associated protein of the present invention 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 substituted amino acid 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 (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).
Said 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 the gene of the said GAP-associated protein GAP MCHR1-C of coding.Concrete, said encoding sox has the nucleotide sequence shown in the SEQ ID NO:2.
Because the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ NO: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 said 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 possibly 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; The polynucleotide of the hybridization of polynucleotide sequence shown in can SEQ NO:2 (have 50% homology at least; 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 according to the invention.Said " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, like 50% (V/V) 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 polynucleotide sequence of the white MCHR1-C of people MCHR1 epitope district's gene expression associated protein according to the 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 clone's with common structure characteristic polynucleotide passage.Sequence dna fragment of the present invention also can use following method to obtain: double chain DNA sequence is separated from genomic dna in (1); (2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of said 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 MCHR1-C of mensuration people's MCHR1 epitope district's gene expression associated protein; (4), detect the protein product of genetic expression through immunological technique or mensuration BA.Aforesaid method can singly be used, but also several different methods combined utilization.
The gene of the present invention that obtains as stated, 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 type 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 a plurality of clones' cDNA sequence need be measured, the cDNA sequence of total length could be spliced.
The invention still further relates to a kind of recombinant vectors that contains said nucleotide sequence.Among the present invention, the polynucleotide sequence of the white MCHR1-C of coding human MCHR1 epitope district's gene expression associated protein can be inserted in the carrier, contains the recombinant vectors of polynucleotide according to the invention with formation." carrier " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral like adenovirus, retrovirus or other carrier.The carrier that is suitable in the present invention includes but not limited to: 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 (Lee and 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 in host, duplicate and stablize, any plasmid and carrier may be used to make up recombinant expression vector.The expression key character of this newspaper of growing flowers 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 the white MCHR1-C of coding human MCHR1 epitope district's gene expression associated protein and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring 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 P of phage
LPromotor; 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.In carrier, inserting enhancer sequence 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.The example that can take is 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 like eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance etc.
Among the present invention; The polynucleotide of the white MCHR1-C of coding human MCHR1 epitope district's gene expression associated protein 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, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like 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 said 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.
Through the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce the white MCHR1-C of people MCHR1 epitope district's 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 MCHR1-C of coding human MCHR1 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 (like temperature displacement or chemically induced), cell is cultivated for some time again.
In step (3), recombinant polypeptide can encapsulate expresses or is secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating through 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, ultrasonication, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
Main points of the present invention have been to provide the aminoacid sequence shown in nucleotide sequence shown in the SEQ NO:1 and the SEQ NO:2; Under the situation of known this nucleotide sequence and aminoacid sequence; The acquisition of this nucleotide sequence and aminoacid sequence; And the acquisition of related vector, host cell, all be conspicuous to those skilled in the art.
The invention still further relates to the application of described GAP-associated protein GAP MCHR1-C in the test kit of preparation detection pigmented dermatosis MCHR1 antibody.
Concrete, said test kit is for detecting the test kit of MCHR1 autoantibody in the patients with vitiligo serum.The people MCHR1 autoantibody level that is detected, can with lay down a definition the importance of people MCHR1 in patients with vitiligo morbidity be used to diagnose the acting autoimmune disorder of people MCHR1.
The invention still further relates to the application of described encoding sox in the test kit that preparation detection people MCHR1 polynucleotide are expressed.
The expression that the polynucleotide of the white MCHR1-C of coding human MCHR1 epitope district's gene expression associated protein can be used for detecting people MCHR1 polynucleotide is whether or at the unconventionality expression of morbid state servant MCHR1 polynucleotide.Dna sequence dna like the white MCHR1-C of coding human MCHR1 polynucleotide epitope district's gene expression associated protein can be used for biopsy specimen is hybridized to judge the expression of people MCHR1.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 sources.Polynucleotide of the present invention a part or all can be used as probe stationary on little array (Microarray) or DNA chip (being called " gene chip " again), be used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.
The sudden change that detects the white MCHR1-C gene of people's MCHR1 epitope district's gene expression associated protein also can be used for the disease of diagnosing people MCHR1 relevant.The form of the white MCHR1-C of people MCHR1 epitope district gene expression associated protein sudden change comprises that the point mutation compared with the dna sequence dna of the white MCHR1-C of normal wild type people MCHR1 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.
The present invention also provides a kind of method that detects MCHR1 autoantibody in the patients with vitiligo serum; Said method comprises: with patients with vitiligo serum is sample; With healthy person serum is contrast, encapsulates elisa plate with the GAP-associated protein GAP MCHR1-C behind the 1 μ g/mL purifying, and 4 ℃ are spent the night; 1 * PBST washing is clapped and is done; The patients serum's incubation that adds dilution; 1 * PBST flushing adds 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 MV greater than healthy serum A value and is judged to the positive.
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: people MCHR1 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 MCHR1 body, for from now on new medicament screen provides the foundation.
(4) description of drawings
Fig. 1 is the plasmid map of prokaryotic expression carrier pGEX-4T-1;
Fig. 2 cuts the result for the enzyme of recombinant expression vector.M:KB ladder Marker; 1:BamH I/XhoI double digestion plasmid pGEX-MCHR1-C; 3: the pcr amplified fragment of goal gene;
Fig. 3 is that the SDS-PAGE of target protein analyzes.1: middle molecular weight protein Marker; 2: the expressing protein of e. coli bl21; The expressing protein of 3:pGEX-4T-1; 4: the expressing protein of no IPTG inductive pGEX-MCHR1-C; The total protein that 5:IPTG induces back pGEX-MCHR1-C to express; The soluble protein that 6:IPTG induces back pGEX-MCHR1-C to express; 7: the purpose purifying protein.
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 expressing protein of 3:pGEX-4T-1; 4: the expressing protein of no IPTG inductive pGEX-MCHR1-C; The total protein that 5:IPTG induces back pGEX-MCHR1-C to express; The soluble protein that 6:IPTG induces back pGEX-MCHR1-C to express; 7: the purpose purifying protein; Fig. 5 is for being antigen with MCHR1-C, and vitiligo autoantibody positive patient serum IgG antibody titre detects.
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
The experimental technique of unreceipted actual conditions among the embodiment; Usually according to people such as normal condition such as Sambrook; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), 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 Dermatology Department of No.3 People's Hospital, Hangzhou City. laboratory.The TaqDNA polysaccharase is purchased the ability lottery industry biotech firm in the Shen, Shanghai; Restriction enzyme, T4DNA ligase enzyme, N,O-Diacetylmuramidase, isopropyl-(IPTG) and middle molecular weight protein matter marker are purchased the bio-engineering corporation in Shanghai.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-TMB (TMB) solution are purchased the Pierce company in the U.S..
Embodiment 1: case is collected
That collects that 1 day to 2009 December of January in 2008 Dermatology Department of No.3 People's Hospital, Hangzhou City. on the 30th outpatient service goes to a doctor does not merge other diseases and patients with vitiligo serum 100 examples of obvious progress was arranged without immunosuppressant treatment, the state of an illness in nearly 3 months; Vulgaris 82 examples (general property 10 examples wherein; Limitation 8 examples, acra property 16 examples, being dispersed in property 33 examples; Being dispersed in property combination acra property 15 examples), segmental pattern 18 examples; Man's 48 examples, women 52 examples, 16～67 years old age, 32 years old mean age, the course of disease 1 month to 20 years, average 3.5 years.Normal human serum 30 examples, wherein male 16 examples, women 14 examples at 18～47 years old age, average 31 years old, are all picked up from the health check-up healthy person.All are collected case and all obtain patient's informed consent signature and the approval of the reason council of No.3 People's Hospital, Hangzhou City..
Embodiment 2: people MCHR1 epitope district gene M CHR1-C's is synthetic
According to bibliographical information people MCHR1 epitope district 51-80; 85-98; 154-158 and 254-260, (the gene pool accession number: nucleotide sequence NM005297.3) carries out synthetic, and introduces BamH I restriction enzyme site at 5 ' end of gene according to people MCHR1 full-length gene; Introduce Xho I restriction enzyme site and termination codon subsequence at 3 ' end, synthetic gene MCHR1-C size is 183bp.This work is accomplished by Nanjing Genscript Biotechnology Co., Ltd..Epitope district gene after synthetic check order be listed as consistent with SEQ ID NO:2.
Embodiment 3: the structure of people MCHR1 epitope district gene M CHR1-C expression vector
Amplification back MCHR1-C fragment is purified, be connected to the BamH I/Xho I site of prokaryotic expression carrier pGEX-4T-2 (being so kind as to give by professor Zhang Chuanxi of insect science institute of Zhejiang University) behind BamHI/xhoI (purchasing in precious biotechnology (Dalian) ltd) double digestion; After T4DNA ligase enzyme (purchasing in precious biotechnology (Dalian) ltd) connects with recombinant plasmid transformed BL21 competent escherichia coli cell in; Screening recombinant conversion, with the recombinant plasmid pGEX-MCHR1-C after the screening through enzyme cut, PCR serves the order-checking of Hai Shenggong company after identifying.The epitope 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 purchases the bio-engineering corporation in Shanghai) to final concentration 1.0mmol/L, 37 ℃ of inducing culture 4～8h collect thalline by ordinary method.With the ultrasonic disruption cell, 4 ℃, 6s/ time * 3 times; 4 ℃ of centrifugal 5min of 12000r/min; Get cleer and peaceful deposition 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 32660Da; Consistent with expection expression product (SEQ ID NO:1) size; Its soluble protein accounts for 60% of purpose fusion rotein, and the GST albumen size of expressing in the positive control is relative molecular mass 26000Da, 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 500 l lavation buffer solutions are washed 3 times, use 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. 3).
Visible by Fig. 3; Negative control BL21 (lane 2) with do not have target protein without IPTG inductive pGEX-MCHR1-C and express (lane 4); After IPTG induced, positive control pGEX-4T-1 had the protein expression (lane 3) of a relative molecular weight 26000, and pGEX-MCHR1-C has the protein expression of relative molecular weight 32660 to express (lane 5); After ultrasonication; PGEX-MCHR1-C expressed proteins part solvable (lane 6), it can pass through the GST purification kit and handle, and obtains purpose purifying protein (lane 7).
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) in the U.S.; Being diluted to 1: 2000 hatches; Two anti-horseradish peroxidase (HRP) the link coupled sheep anti-mouse antibodies (purchasing Santa CruzBiotechnology company) that use in the U.S.; Be diluted to 1: 5000 and hatch, the immunoreation band uses ultra quick luminescent solution (purchasing the Applygen Technologies company in the U.S.) to detect, and experimental result is made public 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 32660Da place, and positive control has a specific band (Fig. 4) at relative molecular mass 26000Da place.
Visible by Fig. 4; Application mouse-anti GST monoclonal antibody can detect the pGEX-4T-1 expressed proteins has a specific band (lane 3) at relative molecular mass 26000 places; IPTG induce total protein that back pGEX-MCHR1-C expresses, soluble protein with and purifying protein a specific band (lane 5～7) is respectively arranged, BL21 expressed proteins (lane 2) and all do not detect specific band (lane 4) without IPTG inductive pGEX-MCHR1-C expressed proteins at relative molecular mass 32660 places.
Embodiment 6: the antigenicity in vitiligo detects and titer determination
Detect the people MCHR1 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.Encapsulate elisa plate with 1g/mL embodiment 4 gained purifying proteins, 4 ℃ are spent the night, and 1 * PBST washing 3 times is clapped and done.The patients serum's incubation 1h that adds 10 times of dilutions, 1 * PBST flushing 3 times adds 1: 37 ℃ of incubation 30min of 5000HR link coupled rabbit anti-human igg enzyme mark binding substances, 1 * PBST flushing 3 times, behind the TMB lucifuge colour developing 30min, 2mol/L H
2SO
4Termination reaction adopts 450nm and 630nm dual wavelength to measure light absorption value.Add 2 times of standard deviations (
) with MV and be judged to the positive greater than healthy serum A value.
The square formation titre is the result show, antigen coated optimum concn is 0.125ug/ml, and two anti-optimum dilution degrees are 1: 5000.In the progressive stages 100 routine patients with vitiligo, between 36 routine serum and the target protein association reaction is arranged, positive rate is 36%.30 normal healthy controls person's serum do not have positive reaction.Patients with vitiligo serum and the normal healthy controls serum OD value of diluting relatively, result at 1: 10 to 1: 10240 show two groups of sample OD values have in this interval significant difference (P＜0.05, Fig. 5).
Visible by Fig. 5, serum to be checked was carried out doubling dilution since 1: 10, between 1: 10240 o'clock patients serum and healthy serum light absorption value, all there is significance poor.
Embodiment 7 (Comparative Examples): compare with existing polypeptide
(J Clin Invest such as Kemp; 2002; 109 (7): 923-930.) research 55 routine patients with vitiligo serum anti MCHR1 antibody positive rate are 16.4%, and He Qiuhao etc. use the GST-MCHR1 purifying protein that contains 1-94 amino acids B cell epitope to be antigen, and anti-MCHR1 autoantibody positive rate is 13.2% (Chinese journal of dermatology in the detection patients with vitiligo serum; 2005,38 (1): 29-31).The present invention is an antigen with the GST-MCHR1 purifying protein of 4 B cell epitopes containing MCHR1; Anti-MCHR1 autoantibody positive rate is 36% in the detection patients with vitiligo serum; The anti-MCHR1 autoantibody that is higher than existing research detects positive rate; This maybe all to come from progressive stage relevant with selected patients serum to be checked, also possibly only comprise 4 specific antigens epi-positions with the purpose purifying protein and not have other non-specific epi-position relevant.
Embodiment 8: with the antigenic joint-detection of existing endochylema
That collects that 1 day to 2010 June of May in 2010 Dermatology Department of No.3 People's Hospital, Hangzhou City. on the 20th outpatient service goes to a doctor does not merge other diseases and patients with vitiligo serum 149 examples of obvious progress was arranged without immunosuppressant treatment, the most of patients state of an illness in nearly 3 months; Vulgaris 120 examples (limitation 20 examples wherein wherein; Acra property 45 examples; Being dispersed in property 50 examples, general property 5 examples), segmental pattern 29 examples; Man's 70 examples, women 79 examples, 16～70 years old age, 28.7 years old mean age, the course of disease 1 month to 28 years, average 3.9 years.All are collected case and all obtain patient's informed consent signature and the approval of the reason council of No.3 People's Hospital, Hangzhou City..
Be antigen with endochylema antigen TYR-C and membrane antigens MCHR1-C respectively, encapsulate elisa plate respectively, detect IgG antibody in the above-mentioned 149 routine patients with vitiligo.The result shows; The IgG antibody positive rate that with TYR-C is Detection of antigen is 41.3%; The IgG antibody positive rate that with MCHR1-C is Detection of antigen is 39.3%, and the total positives rate that the two detected result is united after the statistics is 45.3%, and the positive rate that detects separately is high; Show and use this two kinds of Detection of antigen simultaneously, use wherein a kind of Detection of antigen better effects if separately.
Claims (4)
1. white MCHR1-C of people MCHR1 epitope district's gene expression associated protein, its aminoacid sequence is shown in SEQ ID NO:1.
2. the gene of coding claim 1 said GAP-associated protein GAP MCHR1-C.
3. the application of the described GAP-associated protein GAP MCHR1-C of claim 1 in the test kit of preparation detection pigmented dermatosis MCHR1 antibody.
4. application as claimed in claim 3 is characterized in that said test kit is for detecting the test kit of MCHR1 autoantibody in the patients with vitiligo serum.
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