CN105153312A - Recombinant human autoantigen SSArRNP - Google Patents

Abstract

The invention discloses a recombinant human autoantigen SSArRNP, the antigen core amino acid sequence is successive combination of a peptide shown as a SEQ ID NO: 1, a connection protective peptide and a peptide shown as a SEQ ID NO: 2, and at least one end of the core amino acid sequence is connected with a protective peptide. The recombinant human autoantigen is short in peptide chain length and is easy to express and purify, a relatively large amount of the antigen with relatively high purity can be obtained at one time, the usage cost is low and economic benefit is good. The recombinant antigen contains two or more antigens at the same time, is capable of detecting two or more antinuclear antibodies, and is beneficial for exploitation of ANA detection reagent.

Description

A kind of recombinant human autoantigen SSArRNP

Technical field

The present invention relates to a kind of recombinant human autoantigen.

Background technology

Autoimmune disorder (AutoimmuneDiseases, 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 (Antinuclearantibody, 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(PointOfCareTesting 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:

SRKHRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVE TQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQEL ISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRT CAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFD(SEQIDNO:1)-connect protection peptide-YNPEVLDITEETLHSRFLEGVRNVASVCLQIGYPTVASVPHSIINGYKRVLALSVE TDYTFPLAEKVKAFLADPSAFVAAAPVAAATTAAPAAAAAPAKVEAKEESEESDED MG(SEQIDNO: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: SYPMVQVFDNGSI(SEQIDNO: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: the sequence of wvcaq, C end protection peptide is: fglfd.

As the further improvement of above-mentioned recombinant human autoantigen, especially, the sequence of antigen is: wvcaqsrkhrdhamvpleeaaqeyqeklqvalgelrrkqelaekleveiaikradw kktvetqksrihaefvqqknflveeeqrqlqelekdereqlrilgekeaklaqqsq alqeliseldrrchssalellqeviivlerseswnlkdlditspelrsvchvpglk kmlrtcavhitldpdtanpwlilsedrrqvrlgdtqqsipgneerfdsypmvqvfd ngsiynpevlditeetlhsrflegvrnvasvclqigyptvasvphsiingykrvla lsvetdytfplaekvkafladpsafvaaapvaaattaapaaaaapakveakeesee sdedmgfglfd(SEQIDNO: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:

gaattctgggtctgtgcccaaagccgcaaacaccgcgatcacgctatggtcccgctggaagaagccgctcaggaatatcaagaaaaactgcaagttgccctgggcgaactgcgtcgcaaacaagaactggcagaaaaactggaagtcgaaattgccatcaaacgtgcagattggaagaaaaccgtggaaacgcagaaaagtcgcattcatgcggaatttgtgcagcagaaaaactttctggttgaagaagaacagcgtcaactgcaggaactggaaaaagatgaacgtgaacagctgcgcattctgggcgaaaaagaagccaaactggcacagcaaagtcaagccctgcaggaactgatctccgaactggaccgtcgctgccacagctctgcactggaactgctgcaggaagtcattatcgtgctggaacgcagtgaatcctggaatctgaaagatctggacatcaccagcccggaactgcgttctgtttgccatgtcccgggtctgaagaaaatgctgcgcacgtgtgctgttcacattaccctggatccggacaccgccaacccgtggctgatcctgtctgaagatcgtcgccaagtccgtctgggcgacacccagcaatcaattccgggtaatgaagaacgctttgattcgtatccgatggtgcaggttttcgacaacggcagcatttacaatccggaagtgctggatatcaccgaagaaacgctgcatagccgttttctggaaggcgtgcgcaacgttgcttctgtctgtctgcagatcggttatccgaccgtggcgtcagttccgcactcgattatcaatggttataaacgtgtcctggccctgagtgtggaaaccgattacacgttcccgctggcagaaaaagttaaagcttttctggcggacccgtccgcgttcgttgcggccgcaccggtcgctgcggccaccacggcagctccggcggccgcagctgcgccggccaaagtggaagcgaaagaagaatcggaagaatcggacgaagatatgggttttggtctgtttgactga ctcgag(SEQIDNO: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 52-kDRo/SSA fluorescence immunoassay detected result;

Fig. 5 is antigen rRNP 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 52-kDRo/SSA(GenBank:AAA36581.1, 52-kDRo/SSAribonucleoprotein [Homosapiens]), rRNP(NCBIReferenceSequence:NP_000993.1, 60SacidicribosomalproteinP0 [Homosapiens]), 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 52-kDRo/SSA and rRNP, the structure of its core sequence is SRKHRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVE TQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQEL ISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRT CAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFD(SEQIDNO:1)-connect protection peptide-YNPEVLDITEETLHSRFLEGVRNVASVCLQIGYPTVASVPHSIINGYKRVLALSVE TDYTFPLAEKVKAFLADPSAFVAAAPVAAATTAAPAAAAAPAKVEAKEESEESDED MG(SEQIDNO: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 wvcaqsrkhrdhamvpleeaaqeyqeklqvalgelrrkqelaekleveiaikradw kktvetqksrihaefvqqknflveeeqrqlqelekdereqlrilgekeaklaqqsq alqeliseldrrchssalellqeviivlerseswnlkdlditspelrsvchvpglk kmlrtcavhitldpdtanpwlilsedrrqvrlgdtqqsipgneerfdsypmvqvfd ngsiynpevlditeetlhsrflegvrnvasvclqigyptvasvphsiingykrvla lsvetdytfplaekvkafladpsafvaaapvaaattaapaaaaapakveakeesee sdedmgfglfd(SEQIDNO:4).Conveniently, recombinant human autoantigen of the present invention is designated as SSArRNP.

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 (SEQIDNO:5) is carried out to codon, by artificial synthesis synthetic gene fragment, be connected into pET30a carrier afterwards, build recombinant expression plasmid: pET30a-SSArRNP.

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 0.2mMIPTG, 3: add 0.6mMIPTG, 4: add 1.0mMIPTG

As can be seen from Figure 1, after adding IPTG, expressed recombinant human autoantigen SSArRNP, and when IPTG concentration is 0.2mM, target protein band is maximum, determines that best IPTG induced concentration is 0.2mM.

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, we can learn, when IPTG concentration is 0.2mM, and when bacterium liquid A600 during induction is about 0.4, protein expression best results, adopts this condition to express recombinant human autoantigen SSArRNP 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 the 0.1mol/LNiCl 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, the centrifugal 30min of 12000g, obtains 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 SSArRNP, 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 1.5h, 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 1.5h, 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 LCSAPEB(fluorescin with marked by streptavidin) solution, under 37 DEG C of conditions, hatch 1.5h, remove liquid in hole and dry, by PBST buffer solution for cleaning 4 times;

6) measurement result: every hole adds 100 μ LPBS damping fluids, is placed in by fluorescence microplate in multi-functional microwell plate fluorescence detector, excites with 540nm wavelength light source, detect fluorescence under 590nm filter, 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 52-kDRo/SSA and antigen rRNP can be detected from recombinant human autoantigen SSArRNP, show that recombinant human autoantigen SSArRNP is successfully prepared to possess the potentiality detected for ANA.

<110> Guangzhou Tianbao Songyuan Biology Science & Technology Development Co., Ltd.

<120> recombinant human autoantigen SSArRNP

<130>

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<170>PatentInversion3.5

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SerArgLysHisArgAspHisAlaMetValProLeuGluGluAlaAla

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GlnGluTyrGlnGluLysLeuGlnValAlaLeuGlyGluLeuArgArg

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AlaAspTrpLysLysThrValGluThrGlnLysSerArgIleHisAla

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ProAspThrAlaAsnProTrpLeuIleLeuSerGluAspArgArgGln

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gaattctgggtctgtgcccaaagccgcaaacaccgcgatcacgctatggtcccgctggaa60

gaagccgctcaggaatatcaagaaaaactgcaagttgccctgggcgaactgcgtcgcaaa120

caagaactggcagaaaaactggaagtcgaaattgccatcaaacgtgcagattggaagaaa180

accgtggaaacgcagaaaagtcgcattcatgcggaatttgtgcagcagaaaaactttctg240

gttgaagaagaacagcgtcaactgcaggaactggaaaaagatgaacgtgaacagctgcgc300

attctgggcgaaaaagaagccaaactggcacagcaaagtcaagccctgcaggaactgatc360

tccgaactggaccgtcgctgccacagctctgcactggaactgctgcaggaagtcattatc420

gtgctggaacgcagtgaatcctggaatctgaaagatctggacatcaccagcccggaactg480

cgttctgtttgccatgtcccgggtctgaagaaaatgctgcgcacgtgtgctgttcacatt540

accctggatccggacaccgccaacccgtggctgatcctgtctgaagatcgtcgccaagtc600

cgtctgggcgacacccagcaatcaattccgggtaatgaagaacgctttgattcgtatccg660

atggtgcaggttttcgacaacggcagcatttacaatccggaagtgctggatatcaccgaa720

gaaacgctgcatagccgttttctggaaggcgtgcgcaacgttgcttctgtctgtctgcag780

atcggttatccgaccgtggcgtcagttccgcactcgattatcaatggttataaacgtgtc840

ctggccctgagtgtggaaaccgattacacgttcccgctggcagaaaaagttaaagctttt900

ctggcggacccgtccgcgttcgttgcggccgcaccggtcgctgcggccaccacggcagct960

ccggcggccgcagctgcgccggccaaagtggaagcgaaagaagaatcggaagaatcggac1020

gaagatatgggttttggtctgtttgactgactcgag1056

Claims (10)

1. a recombinant human autoantigen, its core amino acid sequence is the peptide shown in: the peptide shown in SEQIDNO:1-connection protection peptide-SEQIDNO: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: sypmvqvfdngsi(SEQIDNO:3).

5. the recombinant human autoantigen according to claim 1 or 4, is characterized in that: the sequence of N end protection peptide is: the sequence of wvcaq, C end protection peptide is: fglfd.

6. recombinant human autoantigen according to claim 1, is characterized in that: the sequence of antigen for: as shown in SEQIDNO: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 SEQIDNO:5.

10. a preparation method for recombinant human autoantigen, comprise and being expressed by its encoding sequence importing microorganism, purifying obtains afterwards.