CN1952136A - Gene expression product of extro-cellular domain carboxyl end of human thyrotropin receptor, its preparing method and application in enzyme immune technology - Google Patents
Gene expression product of extro-cellular domain carboxyl end of human thyrotropin receptor, its preparing method and application in enzyme immune technology Download PDFInfo
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Abstract
The invention discloses the expressing product of human thyrotropin receptor extracellular domain carboxy-terminal gene and its preparation methods and the application of the product in the enzyme immunoassay technology. The purpose of this invention is to divide TSHR-ecd cDNA into three regions(hTSHR-N terminal, hTSHR-C terminal and hTSHR-M terminal), clone them respectively, especially the hTSHR-C terminal, construct expression vector, express in E.coli, and establish TRAb (including TSAb. TSBAb) ELISA technology taking the protein of this gene engineering fragmentas antigen. The said invention develops novel channel for further research of TSHR antigen determinant and settles foundation for the ultimate transformation into TSAb, TSBAb clinical diagnostic kit.
Description
Technical field
The invention belongs to gene engineering technology field, be carboxyl terminal gene expression product and preparation method thereof in human thyrotropin receptor extracellular region aminoterminal, carboxyl terminal, interlude three fragments and this product enzyme exempt from and luminous enzyme immune technology in application.
Background technology
Autoimmune thyroid disease (AITD) is a class frequently-occurring disease, comprises Graves ' sick (GD), chronic lymphocytic thyroiditis (HT) etc., and its pathogenesis is not clear fully as yet so far.
Thyrotropin receptor (TSHR) is a kind of glycoprotein that exists on follicular epithelial cell (TEC) film, and it is with after thyrotropin (TSH) combines, and regulates the growth, differentiation of TEC and synthetic and discharge the function of Triiodothyronine.On E﹠H factor basis, the body's immunity ANOMALOUS VARIATIONS causes AITD, TSHR is one of main autoantigen of AITD, stimulate body to produce serum thyrotropin receptor antibody (TRAb), this autoantibody belongs to heterogeneous antibody, comprises thyroid stimulating antibody (TSAb) and Tiroidina blocking antibody (TSBAb), and the former simulates the TSH function, stimulate synthetic release of Triiodothyronine for a long time constantly and cause Graves ' disease, the function of appeal is hyperfunction; The latter combines with the TSHR specific site and stops combining and the performance of physiological action of TSH and TSHR, shows as hypothyroidism.TSHR belongs to g protein coupled receptor family, and it is comprised extracellular fragment, striden film section and very short born of the same parents' inner segment three parts by the glycoprotein that 764 amino acid are formed, and hydrophilic extracellular region is made up of 418 amino-acid residues, and this is distinguished by 9 exons codings.The antigenic determinant of TSH, TSAb and TSBAb mainly is distributed in TSHR extracellular fragment (TSHR-ecd), and TSHR-ecd is TSH performance physiological action or the morbific key link of TRAb.Therefore, the antigenic determinant of inquiring into TSHR helps to understand their 26S Proteasome Structure and Function, particularly at the corresponding antigens determinant of different autoantibodies, pathogenesis, AITD diagnosis typing, the curative effect of exploring AITD are judged and even treat all have tangible theory significance, crucial clinical value and good DEVELOPMENT PROSPECT.
Present research thinks that TSAb mainly is incorporated into the aminoterminal of TSHR-ecd (N-end), and it is the important target area of TSAb that the aa22-61 of report TSHR-ecd N-end is arranged; The target site of TSBAb then concentrates on the carboxyl terminal (C-end) of TSHR-ecd, and wherein Cys301, Cys381 and Try390 are TSBAb bonded key positions; Report to TSHR-interlude antigenic determinant is very limited.The association reaction between the antiserum(antisera) that the research of relevant TSHRAb antigenic determinant is at present mainly originated from gene elmination or replacement, synthetic peptide and AITD patients serum and animal model and the result of study of gene engineering expression product, but the above two have become the bottleneck that this field is launched further investigation owing to lack or space conformation that can not correct response hTSHR-ecd and obtain hTSHR albumen as hTSHR proteantigen determinant research material.Because the distribution of TSHR on the follicular epithelial cell film is very rare and extremely unstable, therefore, the TSHR that is difficult to directly to obtain from TEC q.s carries out correlative study; Though TSHR is in the eukaryotic system expression of having succeedd, and this system expression product overcomes the difficult problem of TSHR albumen space conformation, but it yields poorly, technology relative complex, shortcoming that production cost is high, and it expresses output still can't satisfy the research of TSHR antigenic determinant; Comparatively speaking, prokaryotic expression system technology maturation, output height, technology is simple, production cost is low, remains the best means that a large amount of target proteins are obtained in this research.In recent years Chinese scholars also successively uses different expression vectors to go out the hTSHR total length or the extracellular region protein of solubility at expression in escherichia coli, my chamber report in intestinal bacteria successful expression soluble T SHR-ecd albumen, also progressively improve through the output of repeatedly practising expression product.Since on TSHR-ecd, distributing two kinds of autoantibodies epitope, at present still be difficult to distinguish their definite site, after this fragment gene carried out rough separation and expressing, carrying out the result that the institute of antigenic determinant obtains again can synthetic peptide method gained result have more cogency.People such as Japan scholar Akira Nakai once were divided into the TSHR extracellular region two fragments and carried out prokaryotic expression, but up to now, still no-trump hTSHR extracellular region divides the report that three sections (aminoterminal, carboxyl terminal and interlude) expresses.
External at present TRAb detection technique commonly used has two kinds: 1. radioreceptor assay, its shortcoming are can only survey TRAb can not differentiate TSAb and TSBAb.2. biological analysis: can distinguish TSAb and TSBAb, but complicated operation, unsuitable conventional the use.And its commodity medicine box is all very expensive, though domestic have an introduction, can not promote.Thereby create the detection TRAb of China oneself and the new technology that can distinguish to some extent TSAb and TSBAb very necessary.
Summary of the invention
The objective of the invention is to that TSHR-ecd cDNA is divided into three sections (hTSHR-N end, hTSHR-C end and hTSHR-M sections) and carry out respectively, particularly the hTSHR-C end carried out gene clone, expression vector establishment, E.coli prokaryotic expression, and be that antigen is set up TRAb (comprising TSAb, TSBAb) ELISA detection technique further with this genetically engineered fragment albumen.
The objective of the invention is to be achieved through the following technical solutions:
Inventor's thyrotropin receptor extracellular region aminoterminal, carboxyl terminal, interlude gene expression product wherein aminoterminal, carboxyl terminal are the recombinant fragment albumen with thyrotropin receptor protein antigenicity of people.Said hTSHR-C fragment is expressed and is obtained recombinant fragment albumen (TrxFus-hTSHR-C), and the Nucleotide of its cDNA and aminoacid sequence are:
GCT TTT AAG AAT CAG AAG AAA ATC AGA GGA ATC CTT GAG TCC TTG ATG 1000bp
Ala Phe lys Asn Gln lys lys Ile Arg Gly Ile Leu Glu Ser Leu Met 16AA
1 7 13
TGT AAT GAG AGC AGT ATG CAG AGC TTG CGC CAG AGA AAA TCT GTG AAT 1048bp
Cys Asn Glu Ser Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn 32AA
19 25 31
GCC TTG AAT AGC CCC CTC CAC CAG GAA TAT GAA GAG AAT CTG GGT GAC 1096bp
Ala Leu Asn Ser Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp 48AA
37 43
AGC ATT GTT GGG TAC AAG GAA AAG TCC AAG TTC CAG GAT ACT CAT AAC 1144bp
Ser Ile Val Gly Tyr lys Glu lys Ser lys Phe Gln Asp Thr His Asn 64AA
49 55 61
AAC GCT CAT TAT TAC GTC TTC TTT GAA GAA CAA GAG GAT GAG ATC ATT 1192bp
Asn Ala His Tyr Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile 80AA
67 73 79
GGT TTT GGC CAG GAG CTC AAA AAC CCC CAG GAA GAG ACT CTA CAA GCT 1240bp
Gly Phe Gly Gln Glu Leu lys Asn Pro Gln Glu Glu Thr Leu Gln Ala 96AA
85 91
TTT GAC AGC CAT TAT GAC TAC ACC ATA TGT GGG GAC AGT GAA GAC ATG 1288bp
Phe Asp Ser His Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met 112AA
97 103 109
GTG TGT ACC CCC AAG TCC GAT GAG TTC AAC CCG TGT GAA GAC ATA ATG 1336bp
Val Cys Thr Pro lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met 128AA
115 121 127
GGC TAC AAG TTC CTG AGA ATT 1357bp
Gly Tyr lys Phe Leu Arg Ile 135AA
133 。
Human thyrotropin receptor extracellular region aminoterminal, carboxyl terminal, three kinds of expression products of interlude gene exist with the fusion rotein form, the fusion protein molecule amount is respectively 34.4kD, 29.8kD, (wherein recombinant protein is respectively 21.2kD to 30.1kD, 16.6kD, 16.9kD; Trx is 13.2kD).
The preparation method of human thyrotropin receptor extracellular region carboxyl terminal (also comprising aminoterminal, interlude) gene expression product, adopt the TRIzol single stage method from the parathyroid tissue that Graves ' patient operative treatment downcuts, to extract total RNA, synthetic and amplification hTSHR-C end (also having hTSHR-N end, the hTSHR-M section) encoding gene through RT-PCR, directed cloning inserts carrier pET102/D-His.The heat-shocked method changes pET102/hTSHRc (also having pET102/hTSHRn, pET102/hTSHRm) recombinant expression plasmid in the e. coli bl21 (DE3) over to, carries out prokaryotic expression.Expression condition is: with 0.4-1.2mML
-1Isopropylthio β-D-galactoside (IPTG) is an inductor, 25 ℃-37 ℃, induces 1-12 hour.Adopt sex change Ni
2+-NTA affinitive layer purification system purifying through gradient dialysis, renaturation, concentrated, obtains TrxFus-hTSHRc (also having TrxFus-hTSHRn, TrxFus-hTSHRm) recombinant protein.
The present invention explores TSAb on human thyrotropin receptor extracellular region aminoterminal, carboxyl terminal, interlude gene expression product, the TSBAb antigenic determinant distributes, the TrxFus-hTSHRn and the TrxFus-hTSHRc that are rich in TSAb, TSBAb antigenic determinant are made antigen respectively, be applied to set up human serum TRAb (comprising TSAb, TSBAb) enzyme linked immunological absorption (ELISA) detection technique and set up luminous enzyme immune technology.
Positively effect of the present invention:
1, at first the hTSHR extracellular region is divided three sections to carry out gene clone and express and successfully obtained the three sections goal gene and the warm albumen of recombinating thereof at home and abroad.
2, at home and abroad on three segmental gene engineering products, study first TSAb, TSBAb different fragments in conjunction with epi-position (being antigenic determinant) and distribute.TrxFus-hTSHRc and TrxFus-hTSHRn two target proteins have and Tiroidina blocking antibody (TSBAb) and pungency antibody (TSAb) bonded antigenicity, and wherein the former combines more responsively with TSBAb, illustrates that this fragment is that the TSBAb binding site is preponderated.
3, take the lead in utilizing the recombinant protein of hTSHRN, C two ends successfully set up two ELISA methods can detect human serum TRAb and can distinguish TSAb and TSBAb who preponderates, the method sensitivity, special, accurate, stable and tentatively be used for clinical, for the exploitation of AITD etiological diagnosis test kit is got ready.In addition, lay a good foundation for further inquiring into the biotechnological formulation that antigenic determinant and distribution thereof and research is used for the treatment of AITD.
Description of drawings
Fig. 1 is the evaluation of pcr amplification goal gene;
Fig. 2 is that the double digestion of recombinant plasmid pET102-hTSHRn is identified;
Fig. 3 cuts evaluation for the enzyme of recombinant plasmid pET102-hTSHRm;
Fig. 4 cuts evaluation for the enzyme of recombinant plasmid pET102-hTSHRc;
Fig. 5 is the influence of induction time to pET102-TSHRn expressing fusion protein output;
Fig. 6 is the SDS-PAGE electrophoresis of affinitive layer purification product;
Fig. 7 is the protein standard curve;
Fig. 8 is the influence of induction time to expressing fusion protein output;
Fig. 9 is the influence of induction time to expressing fusion protein output;
Figure 10 is that hTSHR-ecd aminoterminal prokaryotic expression protein immunocompetence is identified (Westernblot).
Embodiment
One, the structure and the evaluation of human thyrotropin receptor extracellular region aminoterminal, interlude and carboxyl terminal gene clone, expression plasmid
(1) human thyrotropin receptor extracellular region aminoterminal, interlude and carboxyl terminal gene clone
Employing TRIzol single stage method is synthetic human thyroid cDNA first chain of the total RNA reverse transcription of extraction from the parathyroid tissue of Graves ' patient operative treatment cutting-out, and increasing respectively with the capable RT-PCR of primer that designs voluntarily, hTSHR-N holds, hTSH-C holds and the hTSHR-M encoding gene.With the nucleic acid molecular weight standard is reference, identifies this pcr amplification product with 1.5% agarose gel electrophoresis, the result be presented at 462bp, 417bp and 405bp place visible respectively one clearly, special electrophoretic band, (see figure 1) conforms to expection gene fragment size.Among Fig. 1:
Swimming lane 1:DNA Marker;
Swimming lane 2:PCR hTSHRc amplified production;
Swimming lane 3:PCR hTSHRm amplified production;
Swimming lane 4:PCR hTSHR
nAmplified production.
(2) recombinant expression plasmid pET102-hTSHR
nStructure and evaluation:
With hTSHR
nThe gene directed cloning inserts pET102/D-His carrier, recombinant expression plasmid called after pET102-hTSHR
nThe heat-shocked method is transformed into expresses bacterium E.coli BL21.
1. the enzyme of recombinant plasmid pET102-hTSHRn is cut evaluation:
The pET102-hTSHRn recombinant expression vector is through Pst I single endonuclease digestion, cut the single band of 6770kb; Ssp I enzyme is cut, cut 1105bp and 5669bp two bands, (see figure 2) all is consistent with the Theoretical Calculation result.Among the figure:
1、2：DNA Marker；
3: recombinant plasmid;
4:Pst I enzyme is cut recombinant plasmid;
5:Ssp I enzyme is cut recombinant plasmid.
2. the order-checking of recombinant plasmid pET102-hTSHRn is identified:
With recombinant plasmid pET102-hTSHRn is template, be sequencing primer with TrxFus, T7Reverse respectively, this recombinant plasmid is carried out partial sequence to be measured, measure the sequence that is about 700bp, the result shows the target gene fragment that this carrier is forgiven and Nagayama report sequence is in full accord, direction of insertion is correct, and is correctly identical with the upstream and downstream interface of pET102 carrier.
The Trx restriction enzyme site
TTC CTC GAC GCT AAC CTG GCC GGC TCT GGA TCC GGT
GAT GAC GAT GAC
Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser
Gly Asp Asp Asp Asp
Upstream Interface
AAG CTG GGA ATT GAT CCC TT
C ACC ATG GGG TGT TCG TCT CCA CCC TGC
Lys Leu Gly Ile Asp Pro
Phe Thr Met Gly Cys Ser Ser Pro Pro Cys
GAG TGC CAT CAG GAG GAG GAC TTC AGA GTC ACC TGC AAG GAT ATT CAA
Glu Cys His Gln Glu Glu Asp Phe Arg Val Thr Cys lys Asp Ile Gln
CGC ATC CCC AGC TTA CCG CCC AGT ACG CAG ACT CTG AAG CTT ATT GAG
Arg Ile Pro Ser Leu Pro Pro Ser Thr Gln Thr Leu lys Leu Ile Glu
ACT CAC CTG AGA ACT ATT CCA AGT CAT GCA TTT TCT AAT CTG CCC AAT
Thr His Leu Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn
ATT TCC AGA ATC TAC GTA TCT ATA GAT GTG ACT CTG CAG CAG CTG GAA
Ile Ser Arg Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu
TCA CAC TCC TTC TAC AAT TTG AGT AAA GTG ACT CAC ATA GAA ATT CGG
Ser His Ser Phe Tyr Asn Leu Ser lys Val Thr His Ile Glu Ile Arg
AAT ACC AGG AAC TTA ACT TAC ATA GAC CCT GAT GCC CTC AAA GAG CTC
Asn Thr Arg Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu lys Glu Leu
CCC CAC CTA AAG TTC CTT GGC ATT TTC AAC ACT GGA CTT AAA ATG TTC
Pro His Leu lys Phe Leu Gly Ile Phe Asn Thr Gly Leu lys Met Phe
CCT GAC CTG ACC AAA GTT TAT TCC ACT GAT ATA TTC TTT ATA CTT GAA
Pro Asp Leu Thr lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu
ATT ACA GAC AAC CCT TAC ATG ACG TCA ATC CCT GTG AAT GCT TTT CAG
Ile Thr Asp Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln
Downstream interface
GGA CTA
AAG GGC GAG CTC AAG CTT GAA GGT AAG CCT ATC CCT AAC CCT
Gly Leu
Lys Gly Glu Leu Lys Leu Glu Gly Lys Pro Ile Pro Asn Pro
The 6*His label
CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT
CAT CAT CAC CAT CAC CAT
Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly
His His His His His His
TGA GTT TGA TCC GGC TGC TAA CAA AGC CCG AAA GGA AGC TGA GTT GGC
TGC TGC CAC CGC TGA GCA ATA ACT A
(3) structure of recombinant expression plasmid pET102-hTSHRm and evaluation:
With hTSHR
mThe gene directed cloning inserts pET102/D-His carrier, recombinant expression plasmid called after pET102-hTSHR
mThe heat-shocked method is transformed into expresses bacterium E.coli BL21.
1. the enzyme of recombinant plasmid pET102/hTSHRm is cut evaluation:
The pET102-hTSHRm recombinant expression vector is through Pst I and Stu I after enzyme is cut respectively, all cut the single band of 6770kb; Behind Pst I+Stu I double digestion, cut 1530bp and 5191bp two bands, (seeing shown in Figure 3) all conforms to the Theoretical Calculation result.Among the figure:
1、2：DNA Marker；
3: recombinant plasmid;
4:Pst I+Stu I double digestion recombinant plasmid;
5:Stu I single endonuclease digestion recombinant plasmid;
6:Pst I enzyme is cut recombinant plasmid.
2. the order-checking of recombinant plasmid pET102-hTSHRm is identified:
With recombinant plasmid pET102-hTSHRm is template, with TrxFus is sequencing primer, this recombinant plasmid is carried out partial sequence to be measured, measure and comprise upstream and downstream interface, target gene fragment sequence at the interior 700bp of being about, the result shows that the sequence of reports such as target gene fragment and Nagayama is in full accord, direction of insertion is correct, correctly coincide with pET102 carrier upstream and downstream interface.
The Trx restriction enzyme site
TTC CTC GAC GCT AAC CTG GCC GGC TCT GGA TCC GGT
GAT GAC GAT GAC
Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly
Asp Asp Asp Asp
Upstream Interface
AAG CTG GGA ATT GAT CCC TT
C ACC AAC CCT TAC ATG ACG TCA ATC CCT
Lys Leu Gly Ile Asp Pro Phe Thr Asn Pro Tyr Met Thr Ser Ile Pro
GTG AAT GCT TTT CAG GGA CTA TGC AAT GAA ACC TTG ACA CTG AAG CTG
Val Asn Ala Phe Gln Gly Leu Cys Asn Glu Thr Leu Thr Leu lys Leu
TAC AAC AAC GGC TTT ACT TCA GTC CAA GGA TAT GCT TTC AAT GGG ACA
Tyr Asn Asn Gly Phe Thr Ser Val Gln Gly Tyr Ala Phe Asn Gly Thr
AAG CTG GAT GCT GTT TAC CTA AAC AAG AAT AAA TAC CTG ACA GTT ATT
lys Leu Asp Ala Val Tyr Leu Asn lys Asn lys Tyr Leu Thr Val Ile
GAC AAA GAT GCA TTT GGA GGA GTA TAC AGT GGA CCA AGC TTG CTG GAC
Asp lys Asp Ala Phe Gly Gly Val Tyr Ser Gly Pro Ser Leu Leu Asp
GTG TCT CAA ACC AGT GTC ACT GCC CTT CCA TCC AAA GGC CTG GAG CAC
Val Ser Gln Thr Ser Val Thr Ala Leu Pro Ser lys Gly Leu Glu His
CTG AAG GAA CTG ATA GCA AGA AAC ACC TGG ACT CTT AAG AAA CTT CCA
Leu lys Glu Leu Ile Ala Arg Asn Thr Trp Thr Leu lys lys Leu Pro
CTT TCC TTG AGT TTC CTT CAC CTC ACA CGG GCT GAC CTT TCT TAC CCA
Leu Ser Leu Ser Phe Leu His Leu Thr Arg Ala Asp Leu Ser Tyr Pro
AGC CAC TGC TGT GCT TTT AAG AAT CAG AAG AAA ATC AGA GGA ATC CTT
Ser His Cys Cys Ala Phe lys Asn Gln lys lys Ile Arg Gly Ile Leu
GAG TCC TTG ATG AGC CAC TGC TGT GCT TTT AAG AAT CAG AAG AAA ATC
Glu Ser Leu Met Ser His Cys Cys Ala Phe lys Asn Gln lys lys Ile
Downstream interface
AGA GGA ATC CTT GAG TCC TTG
AAG GGC GAG CTC AAG CTT GAA GGT AAG
Arg Gly Ile Leu Glu Ser Leu
Lys Gly Glu Leu Lys Leu Glu Gly Lys
CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT
CAT
Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly
His
The 6*His label
CAT CAC CAT CAC CAT TGA GTT TGA TCC GGC TGC TAA CAA AGC CCG AAA
His His His His His
GGA AGC TGA GTT GGC TGC TGC CAC CGC TGA GCA ATA ACT A
(4) structure of recombinant expression plasmid pET102-hTSHRc and evaluation:
With hTSHR
cThe gene directed cloning inserts pET102/D-His carrier, recombinant expression plasmid called after pET102-hTSHR
cThe heat-shocked method is transformed into expresses bacterium E.coli BL21.
1. the enzyme of recombinant plasmid pET102/hTSHRc is cut evaluation:
The pET102-hTSHRc recombinant expression vector after Pst I enzyme is cut, cut the single band of 6770kb; Behind Pst I+Ndl I double digestion, cut 1469bp and 5247bp two bands, (seeing shown in Figure 4) is consistent with the Theoretical Calculation result.Among the figure;
1、2：DNA Marker；
3: recombinant plasmid;
4:Pst I single endonuclease digestion recombinant plasmid;
5:Pst I/Ndl I double digestion recombinant plasmid.
2. the order-checking of recombinant plasmid pET102-hTSHRc is identified
With recombinant plasmid pET102-hTSHRc behind the purifying is template, with TrxFus is sequencing primer, this recombinant plasmid is carried out partial sequence to be measured, measure and comprise upstream and downstream interface, target gene fragment sequence at the interior 700bp of being about, the result shows that the sequence of reports such as target gene fragment and Nagayama is in full accord, direction of insertion is correct, correctly coincide with the upstream and downstream interface of pET102 carrier, guaranteed the exactness of single open reading frame.
The Trx restriction enzyme site
TTC CTC GAC GCT AAC CTG GCC GGC TCT GGA TCC GGT
GAT GAC GAT GAC
Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly
Asp Asp Asp Asp
Upstream Interface
AAG CTG GGA ATT GAT CCC TT
C ACC GCT TTT AAG AAT CAG AAG AAA ATC
Lys Leu Gly Ile Asp Pro
Phe Thr Ala Phe lys Asn Gln lys lys Ile
AGA GGA ATC CTT GAG TCC TTG ATG TGT AAT GAG AGC AGT ATG CAG AGC
Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser Ser Met Gln Ser
TTG CGC CAG AGA AAA TCT GTG AAT GCC TTG AAT AGC CCC CTC CAC CAG
Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser Pro Leu His Gln
GAA TAT GAA GAG AAT CTG GGT GAC AGC ATT GTT GGG TAC AAG GAA AAG
Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly Tyr lys Glu lys
TCC AAG TTC CAG GAT ACT CAT AAC AAC GCT CAT TAT TAC GTC TTC TTT
Ser lys Phe Gln Asp Thr His Asn Asn Ala His Tyr Tyr Val Phe Phe
GAA GAA CAA GAG GAT GAG ATC ATT GGT TTT GGC CAG GAG CTC AAA AAC
Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln Glu Leu lys Asn
CCC CAG GAA GAG ACT CTA CAA GCT TTT GAC AGC CAT TAT GAC TAC ACC
Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His Tyr Asp Tyr Thr
ATA TGT GGG GAC AGT GAA GAC ATG GTG TGT ACC CCC AAG TCC GAT GAG
Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro lys Ser Asp Glu
Downstream interface
TTC AAC CCG TGT GAA GAC ATA ATG GGC TAC AAG TTC CTG AGA ATT
AAG
Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr lys Phe Leu Arg Ile
Lys
GGC GAG CTC AAG CTT GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT
Gly Glu Leu Lys Leu Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly
The 6*His label
CTC GAT TCT ACG CGT ACC GGT
CAT CAT CAC CAT CAC CAT TGA GTT TGA
Leu Asp Ser Thr Arg Thr Gly
His His His His His His
TCC GGC TGC TAA CAA AGC CCG AAA GGA AGC TGA GTT GGC TGC
Two, human thyrotropin receptor extracellular region aminoterminal, interlude and carboxyl end groups are because of expression and purifying thereof at prokaryotic system
1. the optimization of abduction delivering condition
At first fixing induction time (all getting 4 hours) adopts different IP TG concentration, and 0.2,0.4,0.5,0.6,0.8 and 1.0mML
-1Fixing IPTG concentration (1.0mML again
-1), adopt different induction times, 1,2,3,4,5 and 6 hour.The results are shown in Figure 5, among the figure:
1-2.pET102/D/LacZ plasmid transforms BL21
TM(DE3), do not induce abduction delivering with IPTG;
3-4.BL21
TM(DE3) empty bacterium is not induced the abduction delivering with IPTG;
5, protein molecular weight sign: 100kD, 75kD, 50kD, 35.0kD, 25.0kD, 15kD;
6-11.IPTG induction time is expressed (abduction delivering 0h, 1h, 2h, 3h, 4h, 5h and 6h) to pET102-TSHRn.
2. affinitive layer purification fusion rotein
The rough mensuration of recombinant plasmid pET102-TSHRn abduction delivering product content in the chromatography product: the SDS-PAGE photo scanning is analyzed, and fusion rotein band optical density(OD) accounts for 91.2% (seeing shown in Figure 6) of total protein band.Among the figure:
1. inclusion body lysate;
2. the inclusion body lysate is crossed post liquid;
3-10. the lavation buffer solution of different steps;
11. protein molecular weight sign: 100kD, 75kD, 50kD, 35kD, 25kD;
12-16. affinitive layer purification after product.
3 molecular weight determinations
By standard protein molecular weight and R thereof
fValue derivation regression equation calculation TrxFus-TSHRn molecular weight is 37.33kD.
4. expression product is quantitative
Set up typical curve (seeing shown in Figure 7) according to the standard protein sample concentration, calculating the Trx-TSHRn productive rate is the 20.6mg/L substratum.Among the figure: y-sample OD
590nm
X-purifying rear fusion protein sample concentration.
(2) expression and purification of hTSHR-ecd interlude gene (hTSHRm) in the prokaryotic hosts bacterium
1. the optimization of abduction delivering condition
At first fixing induction time (all getting 4 hours) adopts different IP TG concentration, and 0.1,0.3,0.5,0.6,0.8 and 1.0mML
-1Fixing IPTG concentration (1.0mML again
-1), adopt different induction times, 1,2,3,4,5 and 6 hour.The results are shown in shown in Figure 8, among the figure:
1, protein molecular weight sign 100kD, 75 kD, 50kD, 35kD, 25kD, 15kD;
2,3, the BL21 that transforms of pET102/D/LacZ plasmid
TM(DE3) induce, abduction delivering not;
4,5, BL21
TM(DE3) empty bacterium is not induced and abduction delivering;
6-12,1.0mML
-1IPTG induces pET102-TSHRm to express (abduction delivering 0h, 1h, 2h, 3h, 4h, 5h and 6h).
2. affinitive layer purification fusion rotein
The SDS-PAGE electrophoresis result of this section affinity chromatography product and the picture analogies of Trx-TSHRn (picture slightly).Through the gel analysis system scan, purpose fusion rotein band optical density(OD) accounts for 92.1% of total protein band optical density(OD) behind the chromatography to the SDS-PAGE photo.
3. molecular weight determination
By standard protein molecular weight and Rf value derivation regression equation TrxFus-TSHRm molecular weight thereof is 30.75D.
4. expression product is quantitative
Check in sample concentration with the protein sample OD value of measuring (after diluting) from this curve, conversing total protein concentration is 42.75mg/dl, and according to the shared per-cent of fusion rotein, calculating the Trx-TSHRm productive rate is the 35.44mg/L substratum.
(3) the hTSHR-ecd carboxyl end groups is because of (hTSHRc) expression and purification in the prokaryotic hosts bacterium
1. the optimization of abduction delivering condition
At first fixing induction time (all getting 5 hours) adopts different IP TG concentration, and 0.2,0.4,0.6,0.8 and 1.0mML
-1Fixing IPTG concentration (1.0mML again
-1), adopt different induction times, 1,2,3,4,5 and 6 hour.The results are shown in shown in Figure 9, among the figure:
1, protein molecular weight sign: 150kD, 100kD, 75kD, 50kD, 35kD, 25kD;
The BL21 that 2-3, pET102/D/LacZ plasmid transform
TM(DE3), do not induce and abduction delivering;
4-5, BL21
TM(DE3) empty bacterium is not induced and abduction delivering;
6-12,1.0mML
-1IPTG abduction delivering (0h, 1h, 2h, 3h, 4h, 5h and 6h).
2. affinitive layer purification fusion rotein
The SDS-PAGE electrophoresis result of this section affinity chromatography product and the picture analogies of Trx-TSHRn (picture slightly).This SDS-PAGE photo scanning is analyzed, and purpose fusion rotein band optical density(OD) accounted for 90.0% of total protein band optical density(OD) after chromatography was tried to achieve in calculating.
3. molecular weight determination
By standard protein molecular weight and R thereof
fValue derivation regression equation.Calculating the TrxFus-TSHRn molecular weight is 28.96kD.
4. expression product is quantitative
Check in sample concentration with the protein sample OD value of measuring (after diluting) from this curve, conversing total protein concentration is 21.83mg/dl, and according to the shared per-cent of fusion rotein, calculating the Trx-TSHRc productive rate is the 28.3mg/L substratum.
Three, the evaluation of human thyrotropin receptor extracellular region aminoterminal, interlude and carboxyl terminal prokaryotic expression protein immunologic competence
(1) adopt Western blot method identifier thyrotropin receptor extracellular region aminoterminal prokaryotic expression protein immunocompetence, step is as follows:
1.SDS-PAGE electrophoresis:
Get purifying TrxTSHRn protein 20 μ l, 100V voltage, SDS-PAGE electrophoresis 4 hours.
2. commentaries on classics film:
1. cut out 6 of 1 of identical nitrocellulose filter of size and gel and 3M filter paper, soak in the water (39mmol/L glycine, 48mmol/L Tris alkali, 0.037%SDS in 6 immersions of 5 minutes Ex-all bubbles and 3M filter paper transfering buffering liquid, 20% methyl alcohol), take out filter paper.
2. the anode of electroporation is down, places 3 layers of 3M filter paper, nitrocellulose filter, gel, 3 layers of 3M filter paper from bottom to top successively, covers cloudy plate.Select electric current, 0.65mA/cm according to the gel area
2, under the room temperature, electrotransfer 2 hours.
3. sealing:
With nitrocellulose filter 20ml rinsing damping fluid (150mmol/L NaCl, 50mmol/LTris.Cl pH7.5) the room temperature rinsing is 10 minutes, repeat 3 times to remove the SDS on the nitrocellulose filter, prevent to influence antibodies afterwards, then film is placed the 5%BSA confining liquid, 4 ℃ of placements of spending the night, with the sealing not with protein bound site.
4. adding one resists:
Discard confining liquid, add positive pooled serum, normal pooled serum respectively, 4 ℃ of smooth placements 2 hours by the amount of every square centimeter of filter membrane 0.2ml.Discard antibody then, with PBS rinsing 3 times, each 10 minutes.
5. goat anti-human igg's two resistive connections with alkali phosphatase enzyme mark close:
Take out nitrocellulose filter and use 20ml rinsing damping fluid room temperature rinsing 10 minutes, abandon clean rinsing liquid, 1: the 500 goat anti-rabbit igg-AP two that presses the amount adding 5%BSA dilution of every square centimeter of filter membrane 0.2ml resists, room temperature was in conjunction with 1 hour, then filter membrane is used 20ml rinsing damping fluid rinsing 3 times, each 10 minutes.
6. colour developing:
Amount by every square centimeter of filter membrane 0.1ml adds chromogenic substrate mixture (66 μ l NBT solution, 10ml alkaline phosphatase damping fluid, 33 μ l BCIP solution), can be observed protein band in 20 minutes, sees shown in Figure 10.
(2) ELISA method identifier thyrotropin receptor extracellular region aminoterminal, interlude and carboxyl terminal prokaryotic expression protein immunocompetence
Three kinds of recombination fusion proteins with purifying are antigen, be one anti-with GD patient and normal human serum respectively, with the goat anti-human igg of horseradish peroxidase-labeled is two anti-, utilize the ELISA reaction conditions of optimizing to measure three sections recombinant protein immunocompetences, with TrxFuS-hTSHRn is antigen, and 38 routine GD patients' OD value is
It is the OD value of 0.59 ± 0.14,20 routine normal controls
Be 0.45 ± 0.08, both have significant difference (P＜0.01), are antigen GD patient OD value with TrxFuS-hTSHRm
Be 0.81 ± 0.40, normal control OD value
Be 1.01 ± 0.46, both there was no significant differences (P＞0.05) are antigen GD patient OD value with TrxFuS-hTSHRc
Be 0.88 ± 0.25, normal control OD value
Be 0.64 ± 0.18, both have significant difference (P＜0.05), and the result shows that TrxFuS-hTSHRn and TrxFuS-hTSHRc can the specificity association reaction take place with the GD patients serum, show that these two fragments have the binding site of TSAb, and the former is more remarkable.
Three kinds of fusion roteins with purifying are antigen equally, with low patients (HT) of 35 routine Hashimoto thyroiditises companion first and 58 routine normal human serums is one to resist, with the goat anti-human igg of alkali phosphorus enzyme labelling is two anti-, utilizes the ELISA reaction conditions of optimizing to measure three sections protein immunization activity, and its result is as follows:
This shows that TrxFuS-hTSHRn and TrxFuS-hTSHRc can the specificity association reaction take place with the HT patients serum, show the binding site of these two fragment tool TSBAb, the latter is more remarkable.
Four, the foundation of the ELISA method of detection TRAb and the preliminary discussion of clinical application:
(1) foundation and the methodology that detects TRAb ELISA method identified, be antigen with TrxFuS-hTSHRn and TrxFuS-hTSHRc respectively, be one anti-with the known TSAb of containing serum and TSBAb serum (positive serum) and normal people's pooled serum (negative serum) respectively, do two with goat anti-human igg's serum of alkali phosphorus enzyme labelling and resist, successfully set up two TRAb ELISA methods.
The schedule of operation of two ELISA methods is basic identical, and is as follows:
The optimization of ELISA condition: carry out condition experiment by intersecting serial dilution, the OD value that records with positive serum is greater than normal control OD value
Or be top condition more than 2 times, the antigen amount of determining ELISA is that TrxFuS-hTSHRn or TrxFuS-hTSHRc are 16ng/100ul, sample serum is dilution in 1: 200, gets 100ul; 100ul is got in the artificial gG serum of alkali phosphorus enzyme labelling goat-anti (two is anti-) dilution in 1: 20000; Developer PNPP1mg/ml gets 100ul; Termination reaction liquid 16mmol/LEDTA or 2N NaOH get 100ul.
With TrxFuS-hTSHRn is that antigenic ELISA methodology is identified: 1) get 63 routine serum, detect TSI through commodity ELISA medicine box and record the result with this method and carry out correlation analysis, as a result correlation coefficient r=0.742P＜0.05 demonstration significant correlation.2) precision: the TSAb sample homogeneous test of getting high, normal, basic content detects 8 samples respectively, and variation within batch cv% is respectively 3.96%, 4.21%, 4.67%; The TSAb sample of getting high, normal, basic content detects respectively 8 times, and batch variation cv% is respectively 7.32%, 7.94%, 8.33%.3) range of normal value: 58 routine normal people's detected results
Be 0.42 ± 0.12, range of normal value is 0.18-0.66
4) detect GD patient's 95 examples
Be 0.90 ± 0.26, be significantly higher than normal control P＜0.01,79 routine OD value＞0.66 wherein, positive rate is 83.16% (79/95).5) detect HT patient's 35 examples
Be 0.66 ± 0.24, be significantly higher than normal control P value＜0.01,21 routine OD value＞0.66 wherein, positive rate 60.0% (21/35).
With TrxFuS-hTSHRc is that antigenic ELISA methodology is identified: 1) precision: the TSBAb sample homogeneous test of getting high, normal, basic content detects 8 samples respectively, variation within batch cv% is respectively 3.24%, 4.63%, 4.72%: the TSBAb sample of getting high, normal, basic content detects respectively 8 times, and batch variation cv% is respectively 7.22%, 8.55%, 8.09%.2) 58 routine normal people's detected results
Be 0.32 ± 0.11, range of normal value is 0.10-0.54
3) detect HT patient's 35 examples
Be 1.06 ± 0.30, be significantly higher than normal control P value＜0.001,33 routine OD value＞0.54 wherein, positive rate 94.2% (33/35).4) detect GD patient's 95 examples
Be 0.73 ± 0.21, be significantly higher than normal control P＜0.01,66 routine OD value＞0.54 wherein, positive rate is 69.4% (66/95).
(2) clinical application: detect normal people's 108 examples altogether, 711 person-times of GD patients, first visit patient 436 examples wherein are with hTSHR
nAnd hTSHR
cTwo ELISA methods setting up for antigen detect every batch sample total positives rate 77.7%-93.3% simultaneously; HT first visit patient 35 examples, two ELISA methods detect every batch sample total positives rate 94.2-97.1% simultaneously; Nontoxic nodular goiter patient 6 examples, total positives rate 0%.
The carboxyl terminal sequence table
＜110〉General Hospital of Tianjin Medical Univ.
＜120〉human thyrotropin receptor extracellular region carboxyl terminal gene expression product and preparation method and the application of this product in enzyme immune technology
<160>1
<210>1
<211>405
<212>DNA
＜213〉Genus Homo (Homo)
<220>
<221>CDS
<222>(953)...(1357)
<400>1
GCT TTT AAG AAT CAG AAG AAA ATC AGA GGA ATC CTT GAG TCC TTG ATG 1000bp
Ala Phe lys Asn Gln lys lys Ile Arg Gly Ile Leu Glu Ser Leu Met 16AA
1 7 13
TGT AAT GAG AGC AGT ATG CAG AGC TTG CGC CAG AGA AAA TCT GTG AAT 1048bp
Cys Asn Glu Ser Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn 32AA
19 25 31
GCC TTG AAT AGC CCC CTC CAC CAG GAA TAT GAA GAG AAT CTG GGT GAC 1096bp
Ala Leu Asn Ser Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp 48AA
37 43
AGC ATT GTT GGG TAC AAG GAA AAG TCC AAG TTC CAG GAT ACT CAT AAC 1144bp
Ser Ile Val Gly Tyr lys Glu lys Ser lys Phe Gln Asp Thr His Asn 64AA
49 55 61
AAG GCT CAT TAT TAC GTC TTC TTT GAA GAA CAA GAG GAT GAG ATC ATT 1192bp
Asn Ala His Tyr Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile 80AA
67 73 79
GGT TTT GGC CAG GAG CTC AAA AAC CCC CAG GAA GAG ACT CTA CAA GCT 1240bp
Gly Phe Gly Gln Glu Leu lys Asn Pro Gln Glu Glu Thr Leu Gln Ala 96AA
85 91
TTT GAC AGC CAT TAT GAC TAC ACC ATA TGT GGG GAC AGT GAA GAC ATG 1288bp
Phe Asp Ser His Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met 112AA
97 103 109
GTG TGT ACC CCC AAG TCC GAT GAG TTC AAC CCG TGT GAA GAC ATA ATG 1336bp
Val Cys Thr Pro lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met 128AA
115 121 127
GGC TAC AAG TTC CTG AGA ATT 1357bp
Gly Tyr lys Phe Leu Arg Ile 135AA
133
Claims (5)
1. a human thyrotropin receptor extracellular region carboxyl end groups is because of (hTSHRc) expression product,
It is characterized in that this hTSHRc fragment is expressed obtains recombinant fragment albumen (TrxFus-hTSHRc), and the Nucleotide of its cDNA and aminoacid sequence are:
GCT TTT AAG AAT CAG AAG AAA ATC AGA GGA ATC CTT GAG TCC TTG ATG 1000bp
Ala Phe lys Asn Gln lys lys Ile Arg Gly Ile Leu Glu Ser Leu Met 16AA
1 7 13
TGT AAT GAG AGC AGT ATG CAG AGC TTG CGC CAG AGA AAA TCT GTG AAT 1048bp
Cys Asn Glu Ser Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn 32AA
19 25 31
GCC TTG AAT AGC CCC CTC CAC CAG GAA TAT GAA GAG AAT CTG GGT GAC 1096bp
Ala Leu Asn Ser Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp 48AA
37 43
AGC ATT GTT GGG TAC AAG GAA AAG TCC AAG TTC CAG GAT ACT CAT AAC 1144bp
Ser Ile Val Gly Tyr lys Glu lys Ser lys Phe Gln Asp Thr His Asn 64AA
49 55 61
AAC GCT CAT TAT TAC GTC TTC TTT GAA GAA CAA GAG GAT GAG ATC ATT 1192bp
Asn Ala His Tyr Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile 80AA
67 73 79
GGT TTT GGC CAG GAG CTC AAA AAC CCC CAG GAA GAG ACT CTA CAA GCT 1240bp
Gly Phe Gly Gln Glu Leu lys Asn Pro Gln Glu Glu Thr Leu Gln Ala 96AA
85 91
TTT GAC AGC CAT TAT GAC TAC ACC ATA TGT GGG GAC AGT GAA GAC ATG 1288bp
Phe Asp Ser His Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met 112AA
97 103 109
GTG TGT ACC CCC AAG TCC GAT GAG TTC AAC CCG TGT GAA GAC ATA ATG 1336bp
Val Cys Thr Pro lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met 128AA
115 121 127
GGC TAC AAG TTC CTG AGA ATT 1357bp
Gly Tyr lys Phe Leu Arg Ile 135AA
133 。
2. human thyrotropin receptor extracellular region carboxyl terminal gene expression product according to claim 1 is characterized in that this expression product is to exist with warm albumen form, and is warm
Albumen hTSHRc molecular weight is 29.8kD, and wherein target protein is 16.6kD, and the Trx molecular weight is 13.2kD.
3. the preparation method of the human thyrotropin receptor extracellular region carboxyl terminal gene expression product of a claim 1, extract total RNA in the parathyroid tissue that it is characterized in that downcutting by Graves ' patient operative treatment, synthetic and amplify hTSHRc end encoding gene through RT-PCR, with the directed cloning technology goal gene is inserted carrier pET102/D-His, with the heat-shocked method with constructed expression vector Transformed E .coli TOP10, screen positive bacterium colony, extracting recombinant expression plasmid pET102/hTSHRc is transformed in the E.coli BL21 prokaryotic expression bacterium, expression condition is an inductor with isopropylthio β-D-galactoside (IPTG), concentration 0.3-1.1mML
-1, 25 ℃-37 ℃ of temperature, time 2-10 hour; Expression product is mainly the inclusion body form, so pass through Ni under the sex change condition
2+The warm albumen of-NTA affinitive layer purification obtains warm albumen TrxFus-hTSHRc through the dialysis renaturation again.
4. the preparation method of expression product according to claim 3 is characterized in that the base sequence of goal gene and expression vector interface is:
AAG CTG GGA ATT GAT CCC TTC ACC GCT TTT AAG AAT CAG。
5. the purposes of the human thyrotropin receptor extracellular region carboxyl terminal gene expression product of claim 1 is characterized in that the hTSHRc end group of hTSHR-ecd gene is applied in enzyme immunoassay technology (ELISA), the chemiluminescence enzyme immunoassay technology (CLEIA) because of expression product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102735850A (en) * | 2012-07-16 | 2012-10-17 | 天津医科大学总医院 | Enzyme-linked immunosorbent assay kit for quantitatively detecting human-serum thyrotrophin receptor antibodies and detecting method |
| CN108761085A (en) * | 2018-05-24 | 2018-11-06 | 成都医学院 | The enzyme linked immunological kit and detection method of thyrotropin receptor parting antibody can be detected simultaneously |
| CN109001472A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
-
2006
- 2006-08-22 CN CNA2006100153820A patent/CN1952136A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102735850A (en) * | 2012-07-16 | 2012-10-17 | 天津医科大学总医院 | Enzyme-linked immunosorbent assay kit for quantitatively detecting human-serum thyrotrophin receptor antibodies and detecting method |
| CN108761085A (en) * | 2018-05-24 | 2018-11-06 | 成都医学院 | The enzyme linked immunological kit and detection method of thyrotropin receptor parting antibody can be detected simultaneously |
| CN109001472A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
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