CN105001341A - Recombinant human autoantigen JoSmD1 - Google Patents
Recombinant human autoantigen JoSmD1 Download PDFInfo
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Abstract
The invention discloses a recombinant human autoantigen. The sequence of antigen core amino acid is peptide showed in SEQ ID NO:1-connection protection peptide-peptide showed in SEQ ID NO:2, and one or more ends of the sequence of the core amino acid are connected with the protection peptide. According to the recombinant human autoantigen, the length of a peptide chain is short, and expression and purification are easy; a great number of high-purity antigen bodies can be obtained in one time, the use cost is low, and the economic benefit is good. The recombinant antigen comprises two or more antigen bodies simultaneously, two or more antinuclear antibodies can be detected simultaneously, and the development of an ANA detection reagent is facilitated.
Description
Technical field
The present invention relates to a kind of recombinant human autoantigen.
Background technology
Autoimmune disorder (Autoimmune Diseases, AID) be general reference immunity of organism effector cell or immune effector molecule, abnormal immune responsing reaction is there is for autologous tissue or cell, cause tissue damage or handicapped disease, that a class sickness rate is high, can whole body system be attacked, serious harm human health, disease that disability rate, lethality rate are high.
In the various test items of AID disease, because antinuclear antibody (ANA) is closely related with AID, be the important target position of autoimmune response, therefore antinuclear antibody detects be in critical positions always in AID disease, is the extremely important shaker test of clinical the next item up.Along with the continuous intensification of people's understanding, the definition of antinuclear antibody (Antinuclear antibody, ANA) expands to present whole cell by originally traditional for nucleus, refers to the general name of the autoantibody of all antigenic components in anti-cell.30 various ANA with different clinical meaning are found at present, common as Anti-hCG action and anti-Sm antibody be the significant antibody of systemic lupus erythematous, anti-SS-A(Ro) antibody and anti-SS-B(La) antibody then more comes across dry syndrome patient etc.
Detection method conventional at present mainly contains two kinds, indirect immunofluorescence (IIF) and ELISA method.IIF method is routine clinical shaker test always, although the method detection sensitivity is high, but need manual operations and microscopic examination, influence factor is more, especially experience does not enrich the easy misjudgment of person, and because the operating time is long, often adopts batch detection clinically, thus also often occur just going out situation about once reporting in one week or two weeks, be unfavorable for the early diagnosis of disease.EILSA method, because carrying out half-quantitative detection, whether the improvement of the clinical frequent judgement of the change according to its concentration patient, thus formulate next step treatment plan, therefore uses widely clinical have also been obtained.Because ELISA method is also batch detection, therefore also exist when specimen amount lacks and can not go out result during pole, test kit length storage period opening packaging easily affects detected result thus affects the shortcoming of clinical diagnosis and treatment.For the problem of above-mentioned clinical existence, POCT(Point Of Care Testing current laboratory medicine development proposes), namely under real-time test frontier background, be devoted to a kind of new detection reagent of exploitation and fluorescence immune chromatography quick detection kit, small fluorescent immunity analysis instrument is used to detect, spended time was at about 15 minutes, therefore simple, convenient, namely sample arrives and namely examines, the problem of patient's waiting time length can be solved, the routing problem that clinician performs autoimmune disorder clinical diagnosis can be solved again, can use at clinical expansion, to improve the treatment level of autoimmune disease.
People's autoantigen is the key point of preparation ANA detection reagent.Natural human autoantigen extraction purification difficulty, be difficult to obtain highly purified sample, cause its use cost high, these problems limit the exploitation of ANA detection reagent.
Summary of the invention
The object of the present invention is to provide a kind of recombinant human autoantigen with good antigenic activity, this antigen has multiple epitope.
The technical solution used in the present invention is:
A kind of recombinant human autoantigen, its core amino acid sequence is:
Gervrglkqqkasaelieenpamtrgryrefyqcdfdiagaeqllqdpklsqnkqa leglgglvgmfdpkgrkvpcvglsigikaellykknpkllnqlqyceeagtplvai igeqelkdgviklrsvtsreevdvrredlveeikrrtg(SEQ ID NO:1)-connect protection peptide-lkavkmtlknrepvqletlsirgnniryfilpdslpldtllvdvepkvkskkreav agrgrgrgrgrgrgrgrgrggprr(SEQ ID NO:2), at least one end of core amino acid sequence is connected with protection peptide.
As the further improvement of above-mentioned recombinant human autoantigen, the length connecting protection peptide is 10 ~ 30 amino acid.Especially, the sequence connecting protection peptide is qplcicsmnth(SEQ ID NO:3).
As the further improvement of above-mentioned recombinant human autoantigen, the length of protection peptide is 4 ~ 10 amino acid.
As the further improvement of above-mentioned recombinant human autoantigen, especially, the sequence of N end protection peptide is: lvklq.
As the further improvement of above-mentioned recombinant human autoantigen, especially, the sequence of antigen is: lvklqgervrglkqqkasaelieenpamtrgryrefyqcdfdiagaeqllqdpkls qnkqaleglgglvgmfdpkgrkvpcvglsigikaellykknpkllnqlqyceeagt plvaiigeqelkdgviklrsvtsreevdvrredlveeikrrtgqplcicsmnthlk avkmtlknrepvqletlsirgnniryfilpdslpldtllvdvepkvkskkreavag rgrgrgrgrgrgrgrgrggprr(SEQ ID NO:4).
A kind of nucleotide sequence, the recombinant human autoantigen that this nucleic acid sequence encoding is above-mentioned.
For improving expression efficiency, nucleotide sequence is the nucleotide sequence having carried out the transformation of inclined preferendum, and the nucleotide sequence having carried out inclined preferendum transformation is:
gatatcctggtgaaactgcaaggtgaacgtgtgcgtggtctgaaacaacaaaaagcaagtgcggaactgattgaagaaaacccggcgatgacgcgtggtcgctatcgtgaattttaccagtgcgatttcgacattgccggtgcggaacagctgctgcaagacccgaaactgtcacagaacaaacaagcactggaaggcctgggcggtctggtgggtatgtttgatccgaaaggccgcaaagttccgtgcgtcggcctgtcgattggtatcaaagctgaactgctgtataagaaaaacccgaaactgctgaatcagctgcaatactgtgaagaagcgggtaccccgctggttgcgattatcggtgaacaggaactgaaagatggcgtgattaaactgcgcagcgttacctctcgtgaagaagtggatgttcgtcgcgaagacctggttgaagaaattaaacgtcgcacgggtcagccgctgtgcatctgtagcatgaacacccatctgaaagcagttaaaatgacgctgaaaaatcgcgaaccggtccaactggaaaccctgagtatccgcggcaacaatattcgttatttcatcctgccggattccctgccgctggacacgctgctggtcgatgtggaaccgaaagtcaaaagtaaaaaacgtgaagcagtggctggccgcggtcgtggtcgtggtcgtggtcgtggtcgtggtcgtggtcgtggtcgtggtggtccgcgtcgttga
ctcgag(SEQ ID NO:5) (black thickened portion is the restriction enzyme site introduced).
The invention has the beneficial effects as follows:
Recombinant human autoantigen of the present invention, peptide chain length is short, is easy to expression and purification.
Recombinant human autoantigen of the present invention, preparation method is simple, can the relatively large and antigen that purity is higher of disposable acquisition, and use cost is low, good in economic efficiency.
Recombinant human autoantigen of the present invention, simultaneously containing plural antigen, can detect two or more antinuclear antibody simultaneously, contribute to the exploitation of ANA detection reagent.
Accompanying drawing explanation
Fig. 1 is recombinant human autoantigen different IP TG concentration abduction delivering result;
Fig. 2 is the different induction starting time expression of results of recombinant human autoantigen;
Fig. 3 is recombinant human autoantigen purified product electrophoresis result;
Fig. 4 is antigen Jo-1 fluorescence immunoassay detected result;
Fig. 5 is antigen SmD1 fluorescence immunoassay detected result.
Embodiment
The effect connecting protection peptide is the antigen peptide that effectively isolation is different, and its length can be arranged as required, general, and its length is 10 ~ 30 amino acid, is preferably 10 ~ 20 amino acid, is more preferred from 10 ~ 15 amino acid.
Protection peptide effect be protect required for antigen peptide not by enzymolysis, be convenient to the operations such as follow-up separation and purification, protection peptide length be generally 4 ~ 10 amino acid.
Below in conjunction with experiment, further illustrate technical scheme of the present invention.
the selection of epitope:
Contriver is according to existing disclosed sequence Jo-1(GenBank:AAX99363 histidyl-tRNA synthetase [Homo sapiens]), SmD1(small nuclear ribonucleoprotein Sm D1 isoform 1 [Homo sapiens] NCBI Reference Sequence:NP_008869.1), epitope is obtained by own method screening, and the aminoacid sequence of epitope peptide concentrated area is intercepted out be stitched together, form two polypeptide chain Jo-1 and SmD1, the structure of its core sequence is: gervrglkqqkasaelieenpamtrgryrefyqcdfdiagaeqllqdpklsqnkqa leglgglvgmfdpkgrkvpcvglsigikaellykknpkllnqlqyceeagtplvai igeqelkdgviklrsvtsreevdvrredlveeikrrtg(SEQ ID NO:1)-connect protection peptide-lkavkmtlknrepvqletlsirgnniryfilpdslpldtllvdvepkvkskkreav agrgrgrgrgrgrgrgrgrggprr(SEQ ID NO:2), at least one end of core amino acid sequence is connected with protection peptide.Then these two polypeptide chains are merged and become a polypeptide chain, be specially: lvklqgervrglkqqkasaelieenpamtrgryrefyqcdfdiagaeqllqdpkls qnkqaleglgglvgmfdpkgrkvpcvglsigikaellykknpkllnqlqyceeagt plvaiigeqelkdgviklrsvtsreevdvrredlveeikrrtgqplcicsmnthlk avkmtlknrepvqletlsirgnniryfilpdslpldtllvdvepkvkskkreavag rgrgrgrgrgrgrgrgrggprr.Conveniently, recombinant human autoantigen of the present invention is designated as JoSmD1.
the structure of recombinant expression plasmid:
Encoding sequence corresponding to above-mentioned two polypeptide chains is found out from the base sequence of people's autoantigen, inclined preferendum transformation (SEQ ID NO:5) is carried out to codon, by artificial synthesis synthetic gene fragment, be connected into pET30a carrier afterwards, build recombinant expression plasmid: pET30a-JoSmD1.
the abduction delivering of recombinant human autoantigen:
1) abduction delivering of different IP TG concentration
Embodiment 1:IPTG concentration is 0.2mM
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to A600 when being 0.4-0.6, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 0.2mM, 20 DEG C of abduction deliverings that spend the night.
Embodiment 2:IPTG concentration is 0.6mM
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to A600 when being 0.4-0.6, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 0.6mM, 20 DEG C of abduction deliverings that spend the night.
Embodiment 3:IPTG concentration is 1.0mM
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to A600 when being 0.4-0.6, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 1.0mM, 20 DEG C of abduction deliverings that spend the night.
Different IP TG concentration abduction delivering result as shown in Figure 1.In Fig. 1, M: albumen Marker, size is followed successively by 130KD from top to bottom, 95KD, 72KD, 55KD, 43KD, 34KD, 26KD, 17KD, 10KD, and 1: namely blank does not add IPTG, 2: add 0.2mM IPTG, 3: add 0.6mM IPTG, 4: add 1.0mM IPTG
As can be seen from Figure 1, after adding IPTG, express recombinant human autoantigen JoSmD1, when IPTG concentration is 0.2mM, target protein band is slightly little, and is 0.6mM in IPTG concentration, during 1.0mM, protein expression does not see change, determines that best IPTG induced concentration is 0.6mM.
2) expression of different induction starting time
Embodiment 4: induction starting time is that bacterium liquid A600 is about 0.4
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to bacterium liquid A600 when being about 0.4, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 1.0mM, 20 DEG C of abduction deliverings that spend the night.
Embodiment 5: induction starting time is that bacterium liquid A600 is about 0.6
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to bacterium liquid A600 when being about 0.6, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 1.0mM, 20 DEG C of abduction deliverings that spend the night.
Embodiment 6: induction starting time is that bacterium liquid A600 is about 0.8
Recombinant expression plasmid is imported e. coli bl21 competent cell, select in the LB liquid nutrient medium that positive transformant is inoculated in containing kantlex, 37 DEG C of shaking table overnight incubation.Next day is inoculated in the fresh substratum of the same race of 300ml by 1:100, and 37 DEG C of shaking tables are cultured to bacterium liquid A600 when being about 0.8, and by bacterium liquid ice bath 30min, then adding IPTG to final concentration is 1.0mM, 20 DEG C of abduction deliverings that spend the night.
The expression of results of different induction starting time as shown in Figure 2.In Fig. 2, M: albumen Marker, size is followed successively by 130KD from top to bottom, 95KD, 72KD, 55KD, 43KD, 34KD, 26KD, 17KD, 10KD, 1: namely blank does not add IPTG, 2: bacterium liquid A600 is about 0.4,3: bacterium liquid A600 is about 0.6,4: bacterium liquid A600 is about 0.8.
As can be seen from Figure 2, along with the increase of bacterial concentration, the target protein stripe size expressed reduces gradually, when showing that bacterium liquid A600 is about 0.4, and protein expression best results.
The qualification result of composition graphs 1 and Fig. 2, can determine: when IPTG concentration is 0.6mM, and when bacterium liquid A600 during induction is about 0.4, protein expression best results, adopts this condition to express recombinant human autoantigen JoSmD1 so unified.
the purifying of recombinant human autoantigen:
By resuspended for the somatic cells appropriate amount of buffer solution after abduction delivering, cell is placed in ice bath ultrasonication, after fragmentation completely, and 4 DEG C, 12000g collected by centrifugation supernatant, purified by nickel ion affinity chromatograph post by supernatant, step is specific as follows:
1) in chromatography column, add 0.1 mol/L NiCl of 3 times of column volumes
2solution, drip clean after add 5 times of column volume distilled waters and rinse, then add 5 times of column volumes of buffer balance nickel ion affinity chromatography posts;
2) supernatant of collection is joined in affinity column, the damping fluid adding 5 times of column volumes only is again dripped until supernatant, then the unconjugated foreign protein of buffer solution elution added containing 50mM imidazoles is clean to wash-out, then collects the target protein after obtaining purifying by the buffer solution elution containing 500mM imidazoles;
3) the target protein solution obtained by purifying loads in dialysis tubing, puts into dialyzate with behind clamp dialysis tubing two ends, 4 DEG C of dialysis 8-10h, middle replacing fresh dialysis fluid 3-4 time.After dialysis, 4 DEG C, centrifugal 30 min of 12000 g, obtain the protein solution after dialysis, for subsequent use.
Recombinant human autoantigen purified product electrophoresis result as shown in Figure 3.In Fig. 3,1: supernatant liquor, 2: with the collection liquid after buffer solution elution one times of column volume, 3: with the collection liquid after 50mM imidazoles wash-out one times of column volume, 4: with 500mM imidazoles wash-out, the 2mL elutriant of collection, 5: with 500mM imidazoles wash-out, the purification of samples (3-7ml elutriant) of collection, 6: with 500mM imidazoles wash-out, the 8ml elutriant of collection, M: albumen Marker, size is followed successively by 130KD from top to bottom, 95KD, 72KD, 55KD, 43KD, 34KD, 26KD, 17KD, 10KD.
As can be seen from Figure 3, adopt nickel ion affinity chromatograph Methods For Purification to arrive recombinant human autoantigen JoSmD1, and antigen purity is higher.
the Activity determination of recombinant human autoantigen:
After obtaining the recombinant human autoantigen after purifying, active with immunofluorence technic detectable antigens, step is specific as follows:
1) fluorescence microplate envelope antigen is used: every hole adds 100 μ L antigenic solutions, and arranges blank (wrapping by PBS damping fluid) and positive control (wrapping by Quality Control antibody), overnight adsorption under 4 DEG C of conditions; Remove liquid in hole after absorption terminates and dry, then using PBST buffer solution for cleaning 4 times;
2) close: every hole adds 200ul confining liquid, hatches 2h, removes liquid in hole and dry, then use PBST buffer solution for cleaning 4 times under 37 DEG C of conditions;
3) primary antibodie is incubated: every hole adds 100 μ L Quality Control antibody, hatches 1.5 h, remove liquid in hole and dry under 37 DEG C of conditions, by PBST buffer solution for cleaning 4 times;
4) add two to resist: every hole adds the biotin labeled mouse-anti human IgG of 100 μ L, hatches 1.5 h, remove liquid in hole and dry under 37 DEG C of conditions, by PBST buffer solution for cleaning 4 times;
5) fluorescin is added: every hole adds 100 μ L CSAPEB(fluorescin with marked by streptavidin) solution, under 37 DEG C of conditions, hatch 1.5 h, remove liquid in hole and dry, by PBST buffer solution for cleaning 4 times;
6) measurement result: every hole adds 100 μ L PBS damping fluids, fluorescence microplate is placed in multi-functional microwell plate fluorescence detector, excite with 540 nm wavelength light sources, under 590 nm filters, detect fluorescence, measure the background value of fluorescence microplate simultaneously.
Recombinant human autoantigen fluorescence immunoassay detected result as shown in Figures 4 and 5.As can be seen from Figure 4 and Figure 5, the activity of antigen Jo-1 and antigen SmD1 can be detected from recombinant human autoantigen JoSmD1, show that recombinant human autoantigen JoSmD1 is successfully prepared to possess the potentiality detected for ANA.
<110> Guangzhou Tianbao Songyuan Biology Science & Technology Development Co., Ltd.
<120> recombinant human autoantigen JoSmD1
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 150
<212> PRT
<213> artificial polypeptide
<400> 1
Gly Glu Arg Val Arg Gly Leu Lys Gln Gln Lys Ala Ser Ala Glu Leu
1 5 10 15
Ile Glu Glu Asn Pro Ala Met Thr Arg Gly Arg Tyr Arg Glu Phe Tyr
20 25 30
Gln Cys Asp Phe Asp Ile Ala Gly Ala Glu Gln Leu Leu Gln Asp Pro
35 40 45
Lys Leu Ser Gln Asn Lys Gln Ala Leu Glu Gly Leu Gly Gly Leu Val
50 55 60
Gly Met Phe Asp Pro Lys Gly Arg Lys Val Pro Cys Val Gly Leu Ser
65 70 75 80
Ile Gly Ile Lys Ala Glu Leu Leu Tyr Lys Lys Asn Pro Lys Leu Leu
85 90 95
Asn Gln Leu Gln Tyr Cys Glu Glu Ala Gly Thr Pro Leu Val Ala Ile
100 105 110
Ile Gly Glu Gln Glu Leu Lys Asp Gly Val Ile Lys Leu Arg Ser Val
115 120 125
Thr Ser Arg Glu Glu Val Asp Val Arg Arg Glu Asp Leu Val Glu Glu
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Ile Lys Arg Arg Thr Gly
145 150
<210> 2
<211> 80
<212> PRT
<213> artificial polypeptide
<400> 2
Leu Lys Ala Val Lys Met Thr Leu Lys Asn Arg Glu Pro Val Gln Leu
1 5 10 15
Glu Thr Leu Ser Ile Arg Gly Asn Asn Ile Arg Tyr Phe Ile Leu Pro
20 25 30
Asp Ser Leu Pro Leu Asp Thr Leu Leu Val Asp Val Glu Pro Lys Val
35 40 45
Lys Ser Lys Lys Arg Glu Ala Val Ala Gly Arg Gly Arg Gly Arg Gly
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Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Pro Arg Arg
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<210> 3
<211> 11
<212> PRT
<213> artificial polypeptide
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Gln Pro Leu Cys Ile Cys Ser Met Asn Thr His
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<210> 4
<211> 246
<212> PRT
<213> artificial polypeptide
<400> 4
Leu Val Lys Leu Gln Gly Glu Arg Val Arg Gly Leu Lys Gln Gln Lys
1 5 10 15
Ala Ser Ala Glu Leu Ile Glu Glu Asn Pro Ala Met Thr Arg Gly Arg
20 25 30
Tyr Arg Glu Phe Tyr Gln Cys Asp Phe Asp Ile Ala Gly Ala Glu Gln
35 40 45
Leu Leu Gln Asp Pro Lys Leu Ser Gln Asn Lys Gln Ala Leu Glu Gly
50 55 60
Leu Gly Gly Leu Val Gly Met Phe Asp Pro Lys Gly Arg Lys Val Pro
65 70 75 80
Cys Val Gly Leu Ser Ile Gly Ile Lys Ala Glu Leu Leu Tyr Lys Lys
85 90 95
Asn Pro Lys Leu Leu Asn Gln Leu Gln Tyr Cys Glu Glu Ala Gly Thr
100 105 110
Pro Leu Val Ala Ile Ile Gly Glu Gln Glu Leu Lys Asp Gly Val Ile
115 120 125
Lys Leu Arg Ser Val Thr Ser Arg Glu Glu Val Asp Val Arg Arg Glu
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Asp Leu Val Glu Glu Ile Lys Arg Arg Thr Gly Gln Pro Leu Cys Ile
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Cys Ser Met Asn Thr His Leu Lys Ala Val Lys Met Thr Leu Lys Asn
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Arg Tyr Phe Ile Leu Pro Asp Ser Leu Pro Leu Asp Thr Leu Leu Val
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<210> 5
<211> 753
<212> DNA
The artificial reconstructed sequence of <213>
<400> 5
gatatcctgg tgaaactgca aggtgaacgt gtgcgtggtc tgaaacaaca aaaagcaagt 60
gcggaactga ttgaagaaaa cccggcgatg acgcgtggtc gctatcgtga attttaccag 120
tgcgatttcg acattgccgg tgcggaacag ctgctgcaag acccgaaact gtcacagaac 180
aaacaagcac tggaaggcct gggcggtctg gtgggtatgt ttgatccgaa aggccgcaaa 240
gttccgtgcg tcggcctgtc gattggtatc aaagctgaac tgctgtataa gaaaaacccg 300
aaactgctga atcagctgca atactgtgaa gaagcgggta ccccgctggt tgcgattatc 360
ggtgaacagg aactgaaaga tggcgtgatt aaactgcgca gcgttacctc tcgtgaagaa 420
gtggatgttc gtcgcgaaga cctggttgaa gaaattaaac gtcgcacggg tcagccgctg 480
tgcatctgta gcatgaacac ccatctgaaa gcagttaaaa tgacgctgaa aaatcgcgaa 540
ccggtccaac tggaaaccct gagtatccgc ggcaacaata ttcgttattt catcctgccg 600
gattccctgc cgctggacac gctgctggtc gatgtggaac cgaaagtcaa aagtaaaaaa 660
cgtgaagcag tggctggccg cggtcgtggt cgtggtcgtg gtcgtggtcg tggtcgtggt 720
cgtggtcgtg gtggtccgcg tcgttgactc gag 753
Claims (10)
1. a recombinant human autoantigen, its core amino acid sequence is the peptide shown in: the peptide shown in SEQ ID NO:1-connection protection peptide-SEQ ID NO:2, and at least one end of core amino acid sequence is connected with protection peptide.
2. recombinant human autoantigen according to claim 1, is characterized in that: the length connecting protection peptide is 10 ~ 30 amino acid.
3. recombinant human autoantigen according to claim 1 and 2, is characterized in that: the length of protection peptide is 4 ~ 10 amino acid.
4. recombinant human autoantigen according to claim 1, is characterized in that: the sequence connecting protection peptide is: qplcicsmnth(SEQ ID NO:3).
5. the recombinant human autoantigen according to claim 1 or 4, is characterized in that: the sequence of N end protection peptide is: LVKLQ.
6. recombinant human autoantigen according to claim 1, is characterized in that: the sequence of antigen for: as shown in SEQ ID NO:4.
7. a nucleotide sequence, is characterized in that: the recombinant human autoantigen described in described nucleic acid sequence encoding claim 1 ~ 6 any one.
8. nucleotide sequence according to claim 7, is characterized in that: nucleotide sequence is the nucleotide sequence having carried out the transformation of inclined preferendum.
9. nucleotide sequence according to claim 7, is characterized in that: nucleotide sequence is as shown in SEQ ID NO:5.
10. a preparation method for recombinant human autoantigen, comprise and being expressed by its encoding sequence importing microorganism, purifying obtains afterwards.
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| CN104334196A (en) * | 2012-02-16 | 2015-02-04 | Atyr医药公司 | Histidyl-trna synthetases for treating autoimmune and inflammatory diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021028337A1 (en) * | 2019-08-09 | 2021-02-18 | Humanitas Mirasole S.P.A. | Peptide antigens and uses thereof |
| CN114729024A (en) * | 2019-08-09 | 2022-07-08 | 博爱米拉索勒股份公司 | Peptide antigens and uses thereof |
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