CN1243097C - Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology - Google Patents
Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology Download PDFInfo
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Abstract
The present invention discloses a gene expression product of a human intercellular adhesion molecule-1 extramembranous I-III functional domain and a preparation method thereof, and an application of the product in radioimmunological technology, which belongs to the technical field of gene engineering. The preparation method comprises the steps that encoding genes of the ICAM-1 extramembranous I-III functional domain are recombined with expression type plasmids pET42a to be converted into prokaryotic expression bacteria, then the mixture is purified to obtain the expression product GST-ICAM-1 which is used as an antigen, and an anti-human polyclonal antibody of rabbits is obtained after the rabbits are immunized. Compared with a eukaryotic system, the expression product of the genes of the human intercellular adhesion molecule-1 extramembranous I-III functional domain in a prokaryotic system has the advantages of simple and convenient operation, easy purification, low cost and high yield. The human serum sICAM-1 radioimmunoassay technology is firstly developed at home and abroad, is primarily applied to clinical detection and has important significance on treatment effect judgment of Graves' disease, medicine withdrawal and recurrence.
Description
Technical field
The invention belongs to gene engineering technology field, specifically is a kind of people's outer I-III function area gene expression product of intercellular adhesion molecule-1 film and preparation method thereof and the application of this product in putting immune technology.
Background technology
Autoimmune thyroid disease is a frequently-occurring disease, comprises Graves ' sick (GD), chronic lymphocytic thyroiditis (HT) etc., and its pathogeny is not clear fully as yet so far.In recent years studies show that intercellular adhesion molecule-1 (intracellular adhesion molecule-1, ICAM-1) with its morbidity closely related.Intercellular adhesion molecule is meant that a group has the glycoprotein material of intercellular adhesion effect at cell surface expression, their participate in cell and cell, cell and extracellular matrix are discerned mutually, adhere mutually, signal transmission and transfer, immunocyte is discerned mutually and interact functions such as leukocytic recirculation and migration.People such as phase early 1990s Frank Schuppert use the scientific discovery of Northern seal stain has ICAM-1 to express in the autoimmune thyroiditis patient, and the normal people expresses very low or do not express.Mirazaki, Moaica M etc. find that with immunohistochemistry technique GD and HT patient's follicular epithelial cell and vessel of thyroid endotheliocyte have ICAM-1 to express.
Graves ' disease is a kind of common autoimmune thyroid disease, and TRAb appears as its Clinical symptoms.Because Graves ' disease is a kind of chronic disease, in therapeutic process, how to estimate result of treatment, to increase, to subtract medicine or drug withdrawal according to the variation of autoantibody in the past.
Think at present because TA can cause producing the autoimmune inflammation in the Tiroidina, lymphocytic infiltration can appear at this position, and lymphocytic autoimmune inflammation position and the endotheliocyte of navigating to of T, the adhesion of target cell, the ICAM-1/LFA-1 adhesive systems plays an important role in the immunologic processes such as interaction of T cell and B cell and antigen presenting cell.
Adhesion molecule (adhesion molecules) is meant to be produced between the iuntercellular be present in the cell surface mediated cell or cell and grey matter by cell and is in contact with one another and the bonded molecule.In the international symposium of the 5th human leukocyte differentiation antigen in 1993, adhesion molecule has been classified as one group of neoantigen separately.Be divided into four classes according to structure, i.e. conglutnin superfamily, immunoglobulin superfamily, selectin family and Cadherin family.
Intercellular adhesion molecule-1 is one of immunoglobulin superfamily adhesion molecule of finding the earliest, is the strand glycoprotein of a cell surface, and the ICAM-1 molecular weight is 80-115KD.On cytolemma, divide extracellular section, TMD and cell inner segment.The mRNA of ICAM-1 contains 1846 bases, 532 amino acid of encoding.Donald in 1988, contain 27 amino acid whose signal attitudes and 505 amino acid whose ICAM-1 albumen in 532 amino acid of reports such as E.S coding, wherein 1-453 encodes, the branch 1-5 extracellular immunoglobulin-like structural area of cell outskirt, be respectively 1-88,89-185,186-284,285-385,386-483.Wherein first immunoglobulin-like structural area combines with LFA-1 (LFA-1) aglucon, participates in antigen recognition; The 3rd immunoglobulin-like structural area combines with complement receptor 3 (Mac-1).ICAM-1 is widely distributed as vascular endothelial cell, thymic epithelial cells, fibroblast, kinds of tumor cells and Tiroidina epithelial cell etc.
Soluble intercellular adhesion molecule-1 (sICAM-1) is the cell surface ICAM-1 formed soluble form that comes off, and molecular weight is about 80KD, and normal human serum concentration is 100-200ng/ml, and sICAM-1 and ICAM-1 have the LFA-1 combined function equally
Its unconventionality expression is to cause one of pathogenetic important factor of Graves '.Compare with other clinical common counter, serum sICAM-1 level is then more quick, sensitive for the situation of its disease of reflection in Graves ' the patient body, so for judging that Graves ' sick curative effect, drug withdrawal and recurrence are significant.
At present, there is no the Tiroidina extract of ICAM-1 both at home and abroad, and the ICAM-1 gene engineering product that obtains through eukaryotic system costs an arm and a leg abroad.
Summary of the invention
The present invention is for solving in the prior art, still the Tiroidina extract that does not have ICAM-1 both at home and abroad, and the utilization genetic engineering technique prepares expression product, and be applied in the Clinical Laboratory, and outer I-III function area gene expression product of the people's who proposes intercellular adhesion molecule-1 film and preparation method thereof and the application of this product in putting immune technology.
The present invention realizes by following technical scheme.
The outer I-III function area gene expression product of a kind of people's intercellular adhesion molecule-1 film, this expression product is the recombinant protein of a kind of people's of having intercellular adhesion molecule-1 protein antigenicity.
Described function area gene expression product, its expression product exists with the fusion rotein form, and the fusion protein molecule amount is 66.37kD, (recombinant protein 31.00kD wherein; Glutathione sulfydryl transferase (glutathione S-transferase GST) 35.37kD).
The preparation method of the outer I-III function area gene expression product of a kind of people's intercellular adhesion molecule-1 film, by the total RNA that extracts in Graves ' the patient parathyroid tissue, synthetic and amplify I-III functional zone encoding gene outside the ICAM-1 film through RT-PCR, be cloned into the pUC18 carrier; The outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid pET42a, recombinant expressed type plasmid is transformed in the E.coli BL21 prokaryotic expression bacterium again, expression condition is: with the 0.5-1% lactose is inductor, 20-37 ℃, induces (3-24) hour; Adopt His.Tag fusion rotein purification system purifying, obtain fusion rotein GST-ICAM-1.
The preparation method of described expression product, the expression condition that its recombinant expressed type plasmid is transformed in the E.coliBL21 expression bacterium can also IPTG0.1-1.0mM be an inductor.
The preparation method of described expression product, the base sequence of its goal gene and expression vector interface is:
CC?ATG?GGA?TAT?CGG
CAG?ACA?TCT?GTG?TCC?CC
The pET42a carrier
Goal gene.
The preparation method of described expression product also can recombinate the outer I-III functional zone of ICAM-1 film encoding gene among the expressive plasmid pET28b, more recombinant expressed type plasmid is transformed among the prokaryotic expression bacterium E.coli BL21.
The preparation method of described expression product can also recombinate the outer I-III functional zone of ICAM-1 film encoding gene among the expressive plasmid pGEX-4T-3, more recombinant expressed type plasmid is transformed among the prokaryotic expression bacterium E.coli BL21.
The outer application of I-III function area gene expression product in putting immune technology of people's intercellular adhesion molecule-1 film, it is characterized in that with expression product GST-ICAM-1 as antigen, immunizing rabbit obtains the anti-H-ICAM-1's of rabbit polyclonal antibody, this antibody is resisted as one to be used for human serum sICAM-1 radiating immuning analysis technology again.
The outer serious karyonide of the expression system of I-III function area gene expression product in protokaryon of such people's intercellular adhesion molecule-1 film is easy and simple to handle, cost is lower, and express product and be easy to purifying, so use prokaryotic expression system to obtain expression product GST-ICAM-1, and then be used for immunizing rabbit as antigen with this, take the lead in developing human serum sICAM-1 radioimmunoassay technique at home and abroad, and Preliminary Applications is in clinical detection, for judging that Graves ' sick curative effect, drug withdrawal and recurrence are significant.Penetrate immunological technique, and Preliminary Applications is in clinical detection, for judging that Graves ' sick curative effect, drug withdrawal and recurrence are significant.
Description of drawings
Fig. 1 is the outer I-III function area gene of RT-PCR amplification H-ICAM-1 film
Fig. 2 cuts for the BglI enzyme and identifies the PCR product
Fig. 3 identifies recombinant plasmid ZpUCICAM for the HindIII single endonuclease digestion
Fig. 4 is that HindIII and EcoRI double digestion are identified recombinant plasmid ZpUCICAM
Fig. 5 identifies recombinant plasmid ZpUCICAM for PCR
Fig. 6 is the interface sequence chart
Fig. 7 is an abduction delivering under the ZpET42aICAM expression vector different condition
Fig. 8 is the comparison of IPTG and lactose-induced expression
Fig. 9 is that fusion rotein GST-ICAM-1 purification result compares
Figure 10 is an Xylene Brilliant Cyanine G albuminimetry typical curve
Figure 11 is recombinant protein GST-ICAM-1Western Blot result
Embodiment
(-), obtain outer I-III function area gene of H-ICAM-1's film and evaluation thereof:
The TRIzol method is extracted the total RNA of Graves patient parathyroid tissue of underwent operative excision, synthetic and the outer I-III function area gene of amplification H-ICAM-1 film of RT-PCR method, with 100bp dna ladder scale standard is the nucleic acid molecular weight standard, 1% agarose gel electrophoresis identifies that PCR product result shows the visible electrophoretic band clearly in about 868bp place, consistent with the target gene fragment size that will increase as Fig. 1, the 1BglI enzyme is cut product among the figure, 2100bp DNALadder.
The BglI enzyme is cut the PCR product, and 1% agarose gel electrophoresis identifies that enzyme cuts the product result, is presented at 505 and visible two electrophoretic bands clearly in 363bp place, is consistent as Fig. 2 with the Theoretical Calculation result, and the 1BglI enzyme is cut product among the figure, 2100bp DNA Ladder.
(2), outer I-III function area gene clone of H-ICAM-1's film and evaluation:
1, recombinant plasmid ZpUCICAM enzyme is cut evaluation:
The outer I-III function area gene of H-ICAM-1's film is inserted into the pUC18 plasmid vector, called after ZpUCICAM, transduction is gone in the DH5 α bacterium.Alkaline lysis method of extracting recombinant plasmid ZpUCICAM, behind the HindIII single endonuclease digestion, the demonstration of 1% agarose gel electrophoresis has an electrophoretic band clearly about the 3554bp place, and contrast pUC18 plasmid is at 2686bp place such as Fig. 3, the 1.HindIII enzyme is cut recombinant plasmid ZpUCICAM among the figure, 2.HindIII enzyme is cut the pUC18 plasmid, 3.15000DL DNA Marker.
HindIII and EcoRI double digestion qualification result are presented at 2631bp and two electrophoretic bands appear in the 923bp place, an electrophoretic band only appears at the 2631bp place and contrast empty pUC18 plasmid, the result is consistent with Theoretical Calculation, as Fig. 4,1 HindIII, EcoRI double digestion recombinant plasmid ZpUCICAM among the figure, 2 HindIII, EcoRI double digestion pUC18 plasmid, 3 15000DL DNA Marker.
2, the PCR of recombinant plasmid ZpUCICAM identifies:
With ZpUCICAM is template, the outer I-III function area gene of pcr amplification H-ICAM-1 film, the result shows the visible electrophoretic band clearly in 868bp place, consistent with the target gene fragment size that will clone, there is not electrophoretic band and contrast empty pUC18 plasmid, as Fig. 5 .1PCR amplification pUC18 product among the figure, 2.PCR amplification ZpUCICAM product, 3.200bp DNALadder.
3, recombinant plasmid ZpUCICAM sequencing analysis:
With recombinant plasmid ZpUCICAM behind the purifying is template, at CEQ2000 type dna sequence analysis instrument the recombinant plasmid that contains I-III function area gene outside H-ICAM-1's film is carried out sequencing, the sequence of report such as measurement result and Donald E.Staunton is in full accord, ICAM-1I-III function area gene and aminoacid sequence (852bp=284aa) (139-990 bit base, 1-284 position AA encodes).The Nucleotide of its cDNA and aminoacid sequence are:
CAG?ACA?TCT?GTG?TCC?CCC?TCA?AAA?GTC?ATC?CTG?CCC?CGG?GGA?GGC?TCC?GTG 189
Gln?Thr?Ser?Val?Ser?Pro?Ser?Lys?Val?Ile?Leu?Pro?Arg?Gly?Gly?Ser?Val 17
CTG?GTG?ACA?TGC?AGC?ACC?TCC?TGT?GAC?CAG?CCC?AAG?TTG?TTG?GGC?ATA?GAG 240
Leu?Val?Thr?Cys?Ser?Thr?Ser?Cys?Asp?Gln?Pro?Lys?Leu?Leu?Gly?Ile?Glu 34
ACC?CCG?TTG?CCT?AAA?AAG?GAG?TTG?CTC?CTG?CCT?GGG?AAC?AAC?CGG?AAG?GTG 291
Thr?Pro?Leu?Pro?Lys?Lys?Glu?Leu?Leu?Leu?Pro?Gly?Asn?Asn?Arg?Lys?Val 51
TAT?GAA?CTG?AGC?AAT?GTG?CAA?GAA?GAT?AGC?CAA?CCA?ATG?TGC?TAT?TCA?AAC 342
Tyr?Glu?Leu?Ser?Asn?Val?Gln?Glu?Asp?Ser?Gln?Pro?Met?Cys?Tyr?Ser?Asn 68
TGC?CCT?GAT?GGG?CAG?TCA?ACA?GCT?AAA?ACC?TTC?CTC?ACC?GTG?TAC?TGG?ACT 393
Cys?Pro?Asp?Gly?Gln?Ser?Thr?Ala?Lys?Thr?Phe?Leu?Thr?Val?Tyr?Trp?Thr 85
CCA?GAA?CGG?GTG?GAA?CTG?GCA?CCC?CTC?CCC?TCT?TGG?CAG?CCA?GTG?GGC?AAG 444
Pro?Glu?Arg?Val?Glu?Leu?Ala?Pro?Leu?Pro?Ser?Trp?Gln?Pro?Val?Gly?Lys 102
AAC?CTT?ACC?CTA?CGC?TGC?CAG?GTG?GAG?GGT?GGG?GCA?CCC?CGG?GCC?AAC?CTC 495
Asn?Leu?Thr?Leu?Arg?Cys?Gln?Val?Glu?Gly?Gly?Ala?Pro?Arg?Ala?Asn?Leu 119
ACC?GTG?GTG?CTG?CTC?CGT?GGG?GAG?AAG?GAG?CTG?AAA?CGG?GAG?CCA?GCT?GTG 546
Thr?Val?Val?Leu?Leu?Arg?Gly?Glu?Lys?Glu?Leu?Lys?Arg?Glu?Pro?Ala?Val 136
GGG?GAG?CCC?GCT?GAG?GTC?ACG?ACC?ACG?GTG?CTG?GTG?AGG?AGA?GAT?CAC?CAT 597
Gly?Glu?Pro?Ala?Glu?Val?Thr?Thr?Thr?Val?Leu?Val?Arg?Ar?Aspg?His?His 153
GGA?GCC?AAT?TTC?TCG?TGC?CGC?ACT?GAA?CTG?GAC?CTG?CGG?CCC?CAA?GGG?CTG 648
Gly?Ala?Asn?Phe?Ser?Cys?Arg?Thr?Glu?Leu?Asp?Leu?Arg?Pro?Gln?Gly?Leu 170
GAG?CTG?TTT?GAG?AAC?ACC?TCG?GCC?CCC?TAC?CAG?CTC?CAG?ACC?TTT?GTC?CTG 699
Glu?Leu?Phe?Glu?Asn?Thr?Ser?Ala?Pro?Tyr?Gln?Leu?Gln?Thr?Phe?Val?Leu 187
CCA?GCG?ACT?CCC?CCA?CAA?CTT?GTC?AGC?CCC?CGG?GTC?CTA?GAG?GTG?GAC?ACG 750
Pro?Ala?Thr?Pro?Pro?Gln?Leu?Val?Ser?Pro?Arg?Val?Leu?Glu?Val?Asp?Thr 204
CAG?GGG?ACC?GTG?GTC?TGT?TCC?CTG?GAC?GGG?CTG?TTC?CCA?GTC?TCG?GAG?GCC 801
Gln?Gly?Thr?Val?Val?Cys?Ser?Leu?Asp?Gly?Leu?Phe?Pro?Val?Ser?Glu?Ala 221
CAG?GTC?CAC?CTG?GCA?CTG?GGG?GAC?CAG?AGG?TTG?AAC?CCC?ACA?GTC?ACC?TAT 852
Gln?Val?His?Leu?Ala?Leu?Gly?Asp?Gln?Arg?Leu?Asn?Pro?Thr?Va?Thr?Tyrl 238
GGC?AAC?GAC?TCC?TTC?TCG?GCC?AAG?GCC?TCA?GTC?AGT?GTG?ACC?GCA?GAG?GAC 903
Gly?Asn?Asp?Ser?Phe?Ser?Ala?Lys?Ala?Ser?Val?Ser?Val?Thr?Ala?Glu?Asp 255
GAG?GGC?ACC?CAG?CGG?CTG?ACG?TGT?GCA?GTA?ATA?CTG?GGG?AAC?CAG?AGC?CAG 954
Glu?Gly?Thr?Gln?Arg?Leu?Thr?Cys?Ala?Val?Ile?Leu?Gly?Asn?Gln?Ser?Gln 272
GAG?ACA?CTG?CAG?ACA?GTG?ACC?ATC?TAC?AGC?TTT?CCG 990
Glu?Thr?Leu?Gln?Thr?Val?Thr?Ile?Tyr?Ser?Phe?Pro 284
(3), the structure and the evaluation of the outer I-III function area gene expression vector of H-ICAM-1's film:
The outer I-III function area gene of people ICAM-I film is inserted among the expression plasmid pET42a called after ZpET42aICAM.
1, recombinant plasmid ZpET42aICAM interface sequence sequencing analysis:
The result shows that recombinant plasmid ZpET42aICAM 5 '-end interface sequence is entirely true, and the amino acid single open reading frame is correct, and as Fig. 6,1 is PET42a carrier 2 among the figure
3 goal gene.
(4), the outer expression of I-III function area gene in intestinal bacteria of H-ICAM-1's film:
The ZpET42aICAM expression vector being transformed into E.coli BL21 expressing in the bacterium, is that inductor carries out abduction delivering with the lactose, and simultaneously with empty bacterium, empty plasmid is contrast.
Expression condition is: with 0.5% lactose is inductor, 20 ℃, induces 6 hours.Get sample on the supernatant liquor behind the ultrasonic broken bacterium, 10%SDS-PAGE result shows that recombinant expression plasmid Transformed E .coli BL21 (DE3) is about the 64.79kD place at molecular weight one protein band of obviously deepening is arranged, and the empty carrier plasmid is about the 35.09kD place at molecular weight one protein band is arranged, empty bacterium does not all have protein band at two places, as Fig. 7, among the figure
Induce 3 hours broken bacterium supernatants of E.coli BL21 (DE3) for 1.0mM IPTG37 ℃
2. induce 3 hours broken bacterium supernatants of pET42a Transformed E .coli BL21 (DE3) for 1.0mM IPTG37 ℃
3. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) for 1.0mM IPTG25 ℃
4. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) for 1.0mM IPTG30 ℃
5. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) for 1.0mM IPTG37 ℃
6. induce 3 hours broken bacterium supernatants of E.coli BL21 (DE3) pLysS for 1.0mM IPTG37 ℃
7. induce 3 hours broken bacterium supernatants of pET42a Transformed E .coli BL21 (DE3) pLysS for 1.0mM IPTG37 ℃
8. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) pLysS for 1.0mM IPTG25 ℃
9. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) pLysS for 1.0mM IPTG30 ℃
10. induce 3 hours broken bacterium supernatants of ZpET42aICAM Transformed E .coli BL21 (DE3) pLysS for 1.0mM IPTG37 ℃
11. low molecular weight protein (LMWP) standard.
(5). the comparison of inductor IPTG and lactose-induced expression
ZpET42aICAM is the optimum expression carrier, with 1.0mMIPTG is inductor, under the optimum expression condition, abduction delivering in the 100ml 2-YT liquid nutrient medium, simultaneously be inductor with 0.5% lactose, 20 ℃ of inducing temperatures, induction time were respectively 6 hours and the expression amount of the comparison target protein that spends the night.The result shows: 1.0mMIPTG, 5% lactose-induced 6 hours and the broken bacterium supernatant target protein that spends the night accounts for overall protein content and is respectively 26.71%, 41.61% and 42.20%.As Fig. 8, among the figure
1. the 0.5% lactose-induced broken bacterium that spends the night is precipitated
2. the 0.5% lactose-induced broken bacterium supernatant that spends the night
3. 0.5% lactose-induced 6 hours broken bacterium are precipitated
4. 0.5% lactose-induced 6 hours broken bacterium supernatants
5. 1.0mMIPTG induces 3 hours broken bacterium precipitations
6. 1.0mMIPTG induces 3 hours broken bacterium supernatants.
(6), the purifying and the evaluation of the outer I-III function area gene expression product of recombinant human ICAM-1 film:
1. the purifying of fusion rotein
Supernatant liquor behind the ultrasonic broken bacterium is respectively with the purification system of His.Tag fusion rotein and the purification system purified fusion protein GST-ICAM-1 of GST.Tag fusion rotein, 10%SDS-PAGE result shows: two kinds of purification systems all can be at the 64.79kD place a visible clear protein band, but also there are other two protein bands in the purification system of GST.Tag fusion rotein, as Fig. 9,1 GST.Tag fusion rotein purification system purified fusion protein GST-ICAM-1 among the figure, 2.His.Tag fusion rotein purification system purified fusion protein GST-ICAM-1,3. low molecular weight protein (LMWP) standard.
The purification system of His.Tag fusion rotein is adopted in decision after relatively.
2, the evaluation of expression product
(1), the mensuration of molecular weight:
According to the molecular weight and the relative mobility thereof of each band of low molecular weight protein (LMWP) standard, set up the linear regression equation of Rf and LgMW, with this regression equation of Rf value substitution of fusion rotein, calculate to such an extent that the fusion protein molecule amount is 66.37kD.
(2), protein quantification:
There is ultraviolet absorption peak at the 595nm place after Xylene Brilliant Cyanine G dyestuff and the protein bound.The purification system purifying gained albumen of His.Tag fusion rotein, microplate reader can be calculated protein concentration, typical curve such as Figure 10 automatically according to the OD value.The concentration of fusion rotein is about 35mg/L.Be converted to protein yield, fusion rotein is about the 35mg/L substratum.
(3), the evaluation of immunologic competence:
Purification of Recombinant GST-ICAM-1 powers on from SDS-PAGE and is transferred on the nitrocellulose filter fully, confirms that through Western Blot recombinant protein has immunocompetence.As Figure 11,1.GST contrast, 2. recombinant protein GST-ICAM-1,3. low molecular weight protein (LMWP) standard among the figure.
(7), the Preliminary Applications of recombinant protein GST-ICAM-1 in clinical detection:
Use the GST-ICAM-1 immunizing rabbit, preparation antibody, as the antibody in the human serum sICAM-1 radioimmunoassay, Preliminary Applications is in clinical detection.Detected result shows: thyroid function is recovered the sick group of normal Graves ' serum sICAM-1 apparently higher than normal group (P＜0.01) after the sick group of the preceding Graves ' of treatment, the pharmacological agent, and the big patient of simple goiter organizes and normal group indifference (P＞0.05); The sick group of Graves ' thyroid function after the pharmacological agent is recovered the sick group of normal Graves ' (P＜0.05) before the treatment; Graves ' patient is before and after the full excision of Tiroidina time, before and after the 131I treatment and before and after the antithyroid drug treatment, shows that through self paired t-test serum sICAM-1 obviously reduces (P＜0.05); The sick end-of-dose failure patient of Graves ' organizes serum sICAM-1 and recovers normal Graves ' patient group (P＜0.01) apparently higher than thyroid function.
Claims (8)
1. the outer I-III function area gene expression product of a people intercellular adhesion molecule-1 film is characterized in that this expression product is the recombinant protein of a kind of people's of having intercellular adhesion molecule-1 protein antigenicity, and the Nucleotide of its cDNA and aminoacid sequence are:
CAG?ACA?TCT?GTG?TCC?CCC?TCA?AAA?GTC?ATC?CTG?CCC?CGG?GGA?GGC?TCC?GTG 189
Gln?Thr?Ser?Val?Ser?Pro?Ser?Lys?Val?Ile?Leu?Pro?Arg?Gly?Gly?Ser?Val 17
CTG?GTG?ACA?TGC?AGC?ACC?TCC?TGT?GAC?CAG?CCC?AAG?TTG?TTG?GGC?ATA?GAG 240
Leu?Val?Thr?Cys?Ser?Thr?Ser?Cys?Asp?Gln?Pro?Lys?Leu?Leu?Gly?Ile?Glu 34
ACC?CCG?TTG?CCT?AAA?AAG?GAG?TTG?CTC?CTG?CCT?GGG?AAC?AAC?CGG?AAG?GTG 291
Thr?Pro?Leu?Pro?Lys?Lys?Glu?Leu?Leu?Leu?Pro?Gly?Asn?Asn?Arg?Lys?Val 51
TAT?GAA?CTG?AGC?AAT?GTG?CAA?GAA?GAT?AGC?CAA?CCA?ATG?TGC?TAT?TCA?AAC 342
Tyr?Glu?Leu?Ser?Asn?Val?Gln?Glu?Asp?Ser?Gln?Pro?Met?Cys?Tyr?Ser?Asn 68
TGC?CCT?GAT?GGG?CAG?TCA?ACA?GCT?AAA?ACC?TTC?CTC?ACC?GTG?TAC?TGG?ACT 393
Cys?Pro?Asp?Gly?Gln?Ser?Thr?Ala?Lys?Thr?Phe?Leu?Thr?Val?Tyr?Trp?Thr 85
CCA?GAA?CGG?GTG?GAA?CTG?GCA?CCC?CTC?CCC?TCT?TGG?CAG?CCA?GTG?GGC?AAG 444
Pro?Glu?Arg?Val?Glu?Leu?Ala?Pro?Leu?Pro?ser?Trp?Gln?Pro?Val?Gly?Lys 102
AAC?CTT?ACC?CTA?CGC?TGC?CAG?GTG?GAG?GGT?GGG?GCA?CCC?CGG?GCC?AAC?CTC 495
Asn?Leu?Thr?Leu?Arg?Cys?Gln?Val?Glu?Gly?Gly?Ala?Pro?Arg?Ala?Asn?Leu 119
ACC?GTG?GTG?CTG?CTC?CGT?GGG?GAG?AAG?GAG?CTG?AAA?CGG?GAG?CCA?GCT?GTG 546
Thr?Val?Val?Leu?Leu?Arg?Gly?Glu?Lys?Glu?Leu?Lys?Arg?Glu?Pro?Ala?Val 136
GGG?GAG?CCC?GCT?GAG?GTC?ACG?ACC?ACG?GTG?CTG?GTG?AGG?AGA?GAT?CAC?CAT 597
Gly?Glu?Pro?Ala?Glu?Val?Thr?Thr?Thr?Val?Leu?Val?Arg?Ar?Aspg?His?His 153
GGA?GCC?AAT?TTC?TCG?TGC?CGC?ACT?GAA?CTG?GAC?CTG?CGG?CCC?CAA?GGG?CTG 648
Gly?Ala?Asn?Phe?Ser?Cys?Arg?Thr?Glu?Leu?Asp?Leu?Arg?Pro?Gln?Gly?Leu 170
GAG?CTG?TTT?GAG?AAC?ACC?TCG?GCC?CCC?TAC?CAG?CTC?CAG?ACC?TTT?GTC?CTG 699
Glu?Leu?Phe?Glu?Asn?Thr?Ser?Ala?Pro?Tyr?Gln?Leu?Gln?Thr?Phe?Val?Leu 187
CCA?GCG?ACT?CCC?CCA?CAA?CTT?GTC?AGC?CCC?CGG?GTC?CTA?GAG?GTG?GAC?ACG 750
Pro?Ala?Thr?Pro?Pro?Gln?Leu?Val?Ser?Pro?Arg?Val?Leu?Glu?Val?Asp?Thr 204
CAG?GGG?ACC?GTG?GTC?TGT?TCC?CTG?GAC?GGG?CTG?TTC?CCA?GTC?TCG?GAG?GCC 801
Gln?Gly?Thr?Val?Val?Cys?Ser?Leu?Asp?Gly?Leu?Phe?Pro?Val?Ser?Glu?Ala 221
CAG?GTC?CAC?CTG?GCA?CTG?GGG?GAC?CAG?AGG?TTG?AAC?CCC?ACA?GTC?ACC?TAT 852
Gln?Val?His?Leu?Ala?Leu?Gly?Asp?Gln?Arg?Leu?Asn?Pro?Thr?Va?Thr?Tyrl 238
GGC?AAC?GAC?TCC?TTC?TCG?GCC?AAG?GCC?TCA?GTC?AGT?GTG?ACC?GCA?GAG?GAC 903
Gly?Asn?Asp?Ser?Phe?Ser?Ala?Lys?Ala?Ser?Val?Ser?Val?Thr?Ala?Glu?Asp 255
GAG?GGC?ACC?CAG?CGG?CTG?ACG?TGT?GCA?GTA?ATA?CTG?GGG?AAC?CAG?AGC?CAG 954
Glu?Gly?Thr?Gln?Arg?Leu?Thr?Cys?Ala?Val?Ile?Leu?Gly?Asn?Gln?Ser?Gln 272
GAG?ACA?CTG?CAG?ACA?GTG?ACC?ATC?TAC?AGC?TTT?CCG 990
Glu?Thr?Leu?Gln?Thr?Val?Thr?Ile?Tyr?Ser?Phe?Pro 284
2. function area gene expression product according to claim 1 is characterized in that this expression product exists with the fusion rotein form, and the fusion protein molecule amount is 66.37kD, wherein recombinant protein 31.00kD; Glutathione sulfydryl transferase 35.37kD.
3. the preparation method of the outer I-III function area gene expression product of a people intercellular adhesion molecule-1 film, it is characterized in that total RNA of extracting by in Graves ' the patient parathyroid tissue, synthetic and amplify I-III functional zone encoding gene outside the ICAM-1 film through RT-PCR, be cloned into the pUC18 carrier; The outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid pET42a, recombinant expressed type plasmid is transformed in the E.coli BL21 prokaryotic expression bacterium again, expression condition is: with the 0.5-1% lactose is inductor, 20-37 ℃, induces 3-24 hour; Adopt His.Tag fusion rotein purification system purifying, obtain fusion rotein GST-ICAM-1.
4. the preparation method of expression product according to claim 3 is characterized in that the expression condition that recombinant expressed type plasmid is transformed in the E.coli BL21 expression bacterium can also IPTG0.1-1.0mM be an inductor.
5. 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:
CC?ATG?GGA?TAT?CGG
GGA?TCC
CAG?ACA?TCT?GTG?TCC?CC
The pET42a carrier
The BamHI siteGoal gene.
6. the preparation method of expression product according to claim 3, it is characterized in that the outer I-III functional zone of ICAM-1 film encoding gene also can be recombinated among the expressive plasmid pET28b, more recombinant expressed type plasmid is transformed among the prokaryotic expression bacterium E.coli BL21.
7. the preparation method of expression product according to claim 3, it is characterized in that the outer I-III functional zone of ICAM-1 film encoding gene can also be recombinated among the expressive plasmid pGEX-4T-3, more recombinant expressed type plasmid is transformed among the prokaryotic expression bacterium E.coli BL21.
8. the outer application of I-III function area gene expression product in putting immune technology of people's intercellular adhesion molecule-1 film.
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