CN105001342A - Recombinant human autoantigen SSB-La - Google Patents
Recombinant human autoantigen SSB-La Download PDFInfo
- Publication number
- CN105001342A CN105001342A CN201510459323.1A CN201510459323A CN105001342A CN 105001342 A CN105001342 A CN 105001342A CN 201510459323 A CN201510459323 A CN 201510459323A CN 105001342 A CN105001342 A CN 105001342A
- Authority
- CN
- China
- Prior art keywords
- lys
- glu
- recombinant human
- human autoantigen
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant human autoantigen SSB-La. The sequence of the antigen core amino acid is showed in SEQ ID NO:1, and one or more ends of the sequence of the core amino acid are connected with 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, the economic benefit is good, the recombinant autigen reaction activity is strong, 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.
The technical solution used in the present invention is:
A kind of recombinant human autoantigen, its core amino acid sequence is:
SEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYISIESAKKFVETPGQKYKETDLLI LFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKIGCLLKVRG AKEGIILFKEKAKEALGKAKDANNGNLQLRNKEVTWEVLEGEVEKEALKKIIEDQQ ESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGAT GPVKRAREETDKEEPASKQQKTEN(SEQ ID NO:1), 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 of protection peptide is 4 ~ 20 amino acid.
As the further improvement of above-mentioned recombinant human autoantigen, especially, the sequence of N end protection peptide is: fnvivealskskaelmei(SEQ ID NO:2), and the sequence of C end protection peptide is: GAGDQ.
As the further improvement of above-mentioned recombinant human autoantigen, especially, the sequence of antigen is:
FNVIVEALSKSKAELMEISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYISIESAKKFVETPGQKYKETDLLILFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKIGCLLKVRGAKEGIILFKEKAKEALGKAKDANNGNLQLRNKEVTWEVLEGEVEKEALKKIIEDQQESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGATGPVKRAREETDKEEPASKQQKTENGAGDQ（SEQ ID NO：3）。
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:
ggatccttcaacgtcatcgtggaagcgctgagcaaatctaaggccgaactgatggaaattagcgaagataaaaccaagatccgtcgcagtccgtccaaaccgctgccggaagttacggatgaatataagaacgacgtgaaaaatcgctctgtttacatttcaatcgaatcggccaaaaagtttgtcgaaaccccgggtcagaaatataaggaaacggacctgctgattctgtttaaagatgactacttcgccaaaaagaacgaagagcgtaagcagaataaagtggaagcaaagctgcgcgctaaacaggaacaagaagcgaaacaaaagctggaagaagatgccgaaatgaagagtctggaagaaaaaattggctgcctgctgaaagtgcgcggcgcaaaagaaggtatcatcctgttcaaggaaaaggcaaaagaagctctgggcaaggcgaaagatgccaacaatggtaacctgcaactgcgtaataaagaagttacctgggaagtcctggaaggcgaagttgaaaaagaagctctgaaaaagattatcgaagatcagcaagaaagcctgaacaagtggaaatctaagggtcgtcgctttaaaggcaagggtaaaggcaataaagcggcccagccgggttcaggcaagggtaaagtccagtttcaaggtaaaaagaccaaattcgcgtcggatgacgaacatgatgaacacgacgaaaacggcgcaaccggtccggtgaaacgtgctcgcgaagaaacggataaagaagaaccggcaagcaagcagcaaaaaacggaaaatggcgctggtgaccagtaa
ctcgag(SEQ ID NO:4) (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, reactive behavior is very strong, contributes 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 recombinant human autoantigen fluorescence immunoassay detected result.
Embodiment
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 ~ 20 amino acid, be preferably 4 ~ 15 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 SSB-La(lupus La protein [Homo sapiens] NCBI Reference Sequence:NP_003133.1), epitope is obtained by own method screening, and the aminoacid sequence of epitope peptide concentrated area is intercepted out be stitched together, the core sequence obtained is: SEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYISIESAKKFVETPGQKYKETDLLI LFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKIGCLLKVRG AKEGIILFKEKAKEALGKAKDANNGNLQLRNKEVTWEVLEGEVEKEALKKIIEDQQ ESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGAT GPVKRAREETDKEEPASKQQKTEN(SEQ ID NO:1), at least one end of core amino acid sequence is connected with protection peptide.In order to express core amino acid sequence better, connect auxiliary sequencel at its two ends, finally for the sequence expressed as shown in SEQ ID NO:3.
the structure of recombinant expression plasmid:
Encoding sequence corresponding to aforementioned polypeptides chain is found out from the base sequence of people's autoantigen, inclined preferendum transformation (SEQ ID NO:4) is carried out to codon, by artificial synthesis synthetic gene fragment, be connected into pET30a carrier afterwards, build recombinant expression plasmid: pET30a-SSB-La.
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, expressed recombinant human autoantigen SSB-La, and when IPTG concentration is 0.6mM, target protein band is slightly large, 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.
Different induction starting time expression of results 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, we can learn, 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 SSB-La 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 SSB-La, 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 Figure 4.As can be seen from Fig. 4, the activity of SSB-La can be detected from recombinant human autoantigen SSB-La, and antigenic activity is very strong, shows that recombinant human autoantigen SSB-La is successfully prepared, possesses the potentiality detected for ANA.
<110> Guangzhou Tianbao Songyuan Biology Science & Technology Development Co., Ltd.
<120> recombinant human autoantigen SSB-La
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 248
<212> PRT
<213> artificial polypeptide
<400> 1
Ser Glu Asp Lys Thr Lys Ile Arg Arg Ser Pro Ser Lys Pro Leu Pro
1 5 10 15
Glu Val Thr Asp Glu Tyr Lys Asn Asp Val Lys Asn Arg Ser Val Tyr
20 25 30
Ile Ser Ile Glu Ser Ala Lys Lys Phe Val Glu Thr Pro Gly Gln Lys
35 40 45
Tyr Lys Glu Thr Asp Leu Leu Ile Leu Phe Lys Asp Asp Tyr Phe Ala
50 55 60
Lys Lys Asn Glu Glu Arg Lys Gln Asn Lys Val Glu Ala Lys Leu Arg
65 70 75 80
Ala Lys Gln Glu Gln Glu Ala Lys Gln Lys Leu Glu Glu Asp Ala Glu
85 90 95
Met Lys Ser Leu Glu Glu Lys Ile Gly Cys Leu Leu Lys Val Arg Gly
100 105 110
Ala Lys Glu Gly Ile Ile Leu Phe Lys Glu Lys Ala Lys Glu Ala Leu
115 120 125
Gly Lys Ala Lys Asp Ala Asn Asn Gly Asn Leu Gln Leu Arg Asn Lys
130 135 140
Glu Val Thr Trp Glu Val Leu Glu Gly Glu Val Glu Lys Glu Ala Leu
145 150 155 160
Lys Lys Ile Ile Glu Asp Gln Gln Glu Ser Leu Asn Lys Trp Lys Ser
165 170 175
Lys Gly Arg Arg Phe Lys Gly Lys Gly Lys Gly Asn Lys Ala Ala Gln
180 185 190
Pro Gly Ser Gly Lys Gly Lys Val Gln Phe Gln Gly Lys Lys Thr Lys
195 200 205
Phe Ala Ser Asp Asp Glu His Asp Glu His Asp Glu Asn Gly Ala Thr
210 215 220
Gly Pro Val Lys Arg Ala Arg Glu Glu Thr Asp Lys Glu Glu Pro Ala
225 230 235 240
Ser Lys Gln Gln Lys Thr Glu Asn
245
<210> 2
<211> 18
<212> PRT
<213> artificial polypeptide
<400> 2
Phe Asn Val Ile Val Glu Ala Leu Ser Lys Ser Lys Ala Glu Leu Met
1 5 10 15
Glu Ile
<210> 3
<211> 271
<212> PRT
<213> artificial polypeptide
<400> 3
Phe Asn Val Ile Val Glu Ala Leu Ser Lys Ser Lys Ala Glu Leu Met
1 5 10 15
Glu Ile Ser Glu Asp Lys Thr Lys Ile Arg Arg Ser Pro Ser Lys Pro
20 25 30
Leu Pro Glu Val Thr Asp Glu Tyr Lys Asn Asp Val Lys Asn Arg Ser
35 40 45
Val Tyr Ile Ser Ile Glu Ser Ala Lys Lys Phe Val Glu Thr Pro Gly
50 55 60
Gln Lys Tyr Lys Glu Thr Asp Leu Leu Ile Leu Phe Lys Asp Asp Tyr
65 70 75 80
Phe Ala Lys Lys Asn Glu Glu Arg Lys Gln Asn Lys Val Glu Ala Lys
85 90 95
Leu Arg Ala Lys Gln Glu Gln Glu Ala Lys Gln Lys Leu Glu Glu Asp
100 105 110
Ala Glu Met Lys Ser Leu Glu Glu Lys Ile Gly Cys Leu Leu Lys Val
115 120 125
Arg Gly Ala Lys Glu Gly Ile Ile Leu Phe Lys Glu Lys Ala Lys Glu
130 135 140
Ala Leu Gly Lys Ala Lys Asp Ala Asn Asn Gly Asn Leu Gln Leu Arg
145 150 155 160
Asn Lys Glu Val Thr Trp Glu Val Leu Glu Gly Glu Val Glu Lys Glu
165 170 175
Ala Leu Lys Lys Ile Ile Glu Asp Gln Gln Glu Ser Leu Asn Lys Trp
180 185 190
Lys Ser Lys Gly Arg Arg Phe Lys Gly Lys Gly Lys Gly Asn Lys Ala
195 200 205
Ala Gln Pro Gly Ser Gly Lys Gly Lys Val Gln Phe Gln Gly Lys Lys
210 215 220
Thr Lys Phe Ala Ser Asp Asp Glu His Asp Glu His Asp Glu Asn Gly
225 230 235 240
Ala Thr Gly Pro Val Lys Arg Ala Arg Glu Glu Thr Asp Lys Glu Glu
245 250 255
Pro Ala Ser Lys Gln Gln Lys Thr Glu Asn Gly Ala Gly Asp Gln
260 265 270
<210> 4
<211> 828
<212> DNA
<213> artificial sequence
<400> 4
ggatccttca acgtcatcgt ggaagcgctg agcaaatcta aggccgaact gatggaaatt 60
agcgaagata aaaccaagat ccgtcgcagt ccgtccaaac cgctgccgga agttacggat 120
gaatataaga acgacgtgaa aaatcgctct gtttacattt caatcgaatc ggccaaaaag 180
tttgtcgaaa ccccgggtca gaaatataag gaaacggacc tgctgattct gtttaaagat 240
gactacttcg ccaaaaagaa cgaagagcgt aagcagaata aagtggaagc aaagctgcgc 300
gctaaacagg aacaagaagc gaaacaaaag ctggaagaag atgccgaaat gaagagtctg 360
gaagaaaaaa ttggctgcct gctgaaagtg cgcggcgcaa aagaaggtat catcctgttc 420
aaggaaaagg caaaagaagc tctgggcaag gcgaaagatg ccaacaatgg taacctgcaa 480
ctgcgtaata aagaagttac ctgggaagtc ctggaaggcg aagttgaaaa agaagctctg 540
aaaaagatta tcgaagatca gcaagaaagc ctgaacaagt ggaaatctaa gggtcgtcgc 600
tttaaaggca agggtaaagg caataaagcg gcccagccgg gttcaggcaa gggtaaagtc 660
cagtttcaag gtaaaaagac caaattcgcg tcggatgacg aacatgatga acacgacgaa 720
aacggcgcaa ccggtccggt gaaacgtgct cgcgaagaaa cggataaaga agaaccggca 780
agcaagcagc aaaaaacgga aaatggcgct ggtgaccagt aactcgag 828
Claims (9)
1. a recombinant human autoantigen, its core amino acid sequence is the peptide shown in SEQ ID NO:1, 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 of protection peptide is 4 ~ 20 amino acid.
3. recombinant human autoantigen according to claim 1, is characterized in that: the length of protection peptide is 4 ~ 15 amino acid.
4. recombinant human autoantigen according to claim 1, is characterized in that: the sequence of N end protection peptide is: FNVIVEALSKSKAELMEI(SEQ ID NO:2).
5. the recombinant human autoantigen according to claim 1 or 4, is characterized in that: the sequence of C end protection peptide is: GAGDQ.
6. a nucleotide sequence, is characterized in that: the recombinant human autoantigen described in described nucleic acid sequence encoding Claims 1 to 5 any one.
7. nucleotide sequence according to claim 6, is characterized in that: nucleotide sequence is the nucleotide sequence having carried out the transformation of inclined preferendum.
8. nucleotide sequence according to claim 7, is characterized in that: nucleotide sequence is as shown in SEQ ID NO:-4.
9. a preparation method for recombinant human autoantigen, comprise and being expressed by its encoding sequence importing microorganism, purifying obtains afterwards.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510459323.1A CN105001342A (en) | 2015-07-29 | 2015-07-29 | Recombinant human autoantigen SSB-La |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510459323.1A CN105001342A (en) | 2015-07-29 | 2015-07-29 | Recombinant human autoantigen SSB-La |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105001342A true CN105001342A (en) | 2015-10-28 |
Family
ID=54374229
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510459323.1A Pending CN105001342A (en) | 2015-07-29 | 2015-07-29 | Recombinant human autoantigen SSB-La |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105001342A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113248616A (en) * | 2021-07-07 | 2021-08-13 | 北京艺妙神州医药科技有限公司 | Chimeric antigen receptor targeting GPC3 and uses thereof |
-
2015
- 2015-07-29 CN CN201510459323.1A patent/CN105001342A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| A.G.TZIOUFAS等: "Fine specificity of autoantibodies to La/SSB:epitope mapping, and characterization", 《CLIN.EXP.IMMUNOL.》 * |
| ATHANASIOS G TZIOUFAS等: "Autoantibodies to La/SSB in patients with primary sjogren"s syndrome(pSS) are associated with upregulation of LA/SSB mRNA in minor salivary gland biopsies(MSGs)", 《JOURNAL OF ANTOIMMUNITY》 * |
| KUMAR A等: "Lupus La protein[homo sapiens],登录号：NP_003133.1", 《GENBANK》 * |
| M.HUANG等: "Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B(La) autoantigen in sera from patients with primary sjogren"s syndrome", 《CLINICAL & EXPERIMENTAL IMMUNOLOGY》 * |
| 邓安梅 等: "自身抗原SSB/La抗原优势表位分析", 《中国免疫学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113248616A (en) * | 2021-07-07 | 2021-08-13 | 北京艺妙神州医药科技有限公司 | Chimeric antigen receptor targeting GPC3 and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111217920B (en) | N-S dominant epitope fusion protein of new coronavirus, preparation method and application thereof, expression protein, microorganism, application thereof and kit | |
| CN103497252B (en) | For the single domain heavy chain antibody L5-78 of Listeria monocytogenes | |
| CN107344968B (en) | Time-resolved fluorescence immunoassay method for detecting avian influenza virus H7N9 | |
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN101525614A (en) | Human oophoroma tumor marker HE4 enzymoimmunoassay kit | |
| CN110551213B (en) | Anti-filovirus monoclonal neutralizing antibody and preparation method and application thereof | |
| CN102887943A (en) | B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof | |
| CN102439447A (en) | Method for detecting substance in biological sample | |
| CN111647055A (en) | N protein for detecting novel coronavirus, preparation and application thereof | |
| CN104610443B (en) | A kind of high stability restructuring Procalcitonin, Preparation method and use | |
| CN107304231B (en) | Mycobacterium tuberculosis fusion protein and application thereof | |
| CN109142738A (en) | Marker and its application of the ECM1 as Serologic detection liver fibrosis | |
| CN103389380B (en) | The double-antibody sandwich elisa detection method of a kind of goose FSH | |
| CN113388039B (en) | Antigen mimic epitope of SARS-COV-2 coronavirus and immunochromatography test strip | |
| CN109111507A (en) | Viral recombinant glycoprotein and its eukaryocyte high-efficiency expression method and application | |
| CN105001342A (en) | Recombinant human autoantigen SSB-La | |
| CN105001341A (en) | Recombinant human autoantigen JoSmD1 | |
| CN108530521A (en) | Recombinate the preparation and application of hepatitis C antigen | |
| CN110066343A (en) | A kind of recombinant antigen and its application for detecting HIV new infections | |
| CN114773460A (en) | Nano antibody of targeted rotavirus protein and application thereof | |
| CN105294849B (en) | Spermatogonial stem cells into mouse specific antigen 4933427D06RIK and its antibody and application | |
| CN105061578A (en) | Recombinant human autoantigen Scl-70 | |
| CN105153312A (en) | Recombinant human autoantigen SSArRNP | |
| JPH06511151A (en) | Immunoassay to identify alcoholics and monitor alcohol consumption | |
| CN103497251B (en) | For the single domain heavy chain antibody L5-79 of Listeria monocytogenes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151028 |
|
| RJ01 | Rejection of invention patent application after publication |