CN1448509A - 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
- Publication number
- CN1448509A CN1448509A CN 02103820 CN02103820A CN1448509A CN 1448509 A CN1448509 A CN 1448509A CN 02103820 CN02103820 CN 02103820 CN 02103820 A CN02103820 A CN 02103820A CN 1448509 A CN1448509 A CN 1448509A
- Authority
- CN
- China
- Prior art keywords
- icam
- expression product
- expression
- plasmid
- film
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to gene engineering technology. The I-III functional encoding gene outside intercellular adhesion molecule-1 (ICAM-1) membrane is recombined to expression plasmid pET42a, transferred into prokaryotic expression bacteria, and purified to obtain expression product GST-ICAM-1. The expression product is used as antigen in immunizing rabbit to obtain rabbit anti-human polyclonal antibody. Thus prepared expression product of human I-III functional encoding gene outside ICAM-1 membrane in prokaryotic system is easy to purify, low in cost and high in yield. The human serum sICAM-1 radioimmunoassay technology is developed and used in clinical test and has important significance in diagnosing the treating effect of Graves' disease.
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, Monica 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 (s ICAM-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 can substitute the nucleotide sequence of people's intercellular adhesion molecule-1 total length, its recombinant protein has immunocompetence, and its fusion protein molecule amount is 66.37kD, and productive rate is about the 35mg/L substratum.
Described function area gene expression product, the recombinant plasmid called after ZpUCICAM of its extraction, and its gene fragment size is consistent with want clone gene fragment.
Described function area gene expression product, the recombinant plasmid called after ZpET42aICM of its function area gene expression vector, and its interface sequence, amino acid single open reading frame are:
CC ATG GGA TAT CGG CAG ACA TCT GTG TCC CCThe PET42a carrier
Goal gene.
The preparation method of the outer I-III function area gene expression product of a kind of people's intercellular adhesion molecule-1 film, be the total RNA that extracts 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, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium; In the affinity purification post, purifying obtains expression product GST-ICAM-1, and is multiple through affinitive layer purification, obtains the outer I-III functional zone fusion rotein GST-ICAM-1 of reorganization ICAM-1 film.
The preparation method of described expression product, its recombinant expressed type plasmid is transformed in the E.coli BL21 expression bacterium and expresses.
The preparation method of described expression product, the expression condition that its recombinant expressed type plasmid is transformed in the E.coliBL21 expression bacterium is: with the 0.5-1% lactose is inductor, 20-37 ℃, induces 3-24 hour.
The preparation method of described expression product is to be with the expression condition that recombinant expressed type plasmid is transformed in the E.coli BL21 expression bacterium: be inductor with IPTG0.1-1.0mM.
The preparation method of described expression product is that the outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid PET28b, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium.
The preparation method of described expression product is that the outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid PGEX4T3, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium.
The outer application of I-III function area gene expression product in putting immune technology of people's intercellular adhesion molecule-1 film, be as antigen with expression product GST-ICAM-1, obtain the anti-people's of rabbit polyclonal antibody behind the immunizing rabbit, again with the anti-people's of rabbit polyclonal antibody as an anti-human serum sICAM-1 radiating immuning analysis technology that is used for.
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.
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 Bgl I 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-1 Western Blot result
Embodiment
(1), 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. 1,1 Bgl I enzyme is cut product among the figure, 2 100bp DNALadder.
Bgl I 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 1 Bgl I enzyme is cut product among the figure, 2 100bp 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. 3,1 HindIII enzyme is cut recombinant plasmid ZpUCICAM among the figure, 2 HindIII enzymes are 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 products, 3 200bp DNA Ladder.
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, as follows:
ICAM-1 I-III ( 852bp＝284aa ) CAG ACA TCT GTG TCC CCC TCA AAA GTC ATC CTG CCC CGG GGA GGC TCC GTGGln Thr Ser Val Ser Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Ser ValCTG GTG ACA TGC AGC ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAGLeu Val Thr Cys Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile GluACC CCG TTG CCT AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTGThr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys ValTATGAA CTG AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AACTyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser AsnTGC CCT GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACTCys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp ThrCCA GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TGG CAG CCA GTG GGC AAGPro Glu Arg Val Glu Leu Ala Pro Leu Pro ser Ttp Gln Pro Val Gly LysAAC CTT ACC CTA CGC TGC CAG GTG GAG GGT GGG GCA CCC CGG GCC AAC CTCAsn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn LeuACC GTG GTG CTG CTC CGT GGG GAG AAG GAG CTG AAA CGG GAG CCA GCT GTG Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu Lys Arg Glu Pro Ala ValGGG GAG CCC GCT GAG GTC ACG ACC ACG GTG CTG GTG AGG AGA GAT CAC CATGly Glu Pro Ala Glu Val Thr Thr Thr Val Leu Va1 Arg Ar Aspg His HisGGA GCC AAT TTC TCG TGC CGC ACT GAA CTG GAC CTG CGG CCC CAA GGG CTGGly Ala Asn Phe Ser Cys Arg Thr Glu Leu Asp Leu Arg Pro Gln Gly LeuGAG CTG TTT GAG AAC ACC TCG GCC CCC TAC CAG CTC CAG ACC TTT GTC CTGGlu Leu Phe Glu Asn Thr Ser Ala Pro Tyr Gln Leu Gln Thr Phe Val LeuCCA GCG ACT CCC CCA CAA CTT GTC AGC CCC CGG GTC CTA GAG GTG GAC ACGPro Ala Thr Pro Pro Gln Leu Val Ser Pro Arg Val Leu Glu Val Asp ThrCAG GGG ACC GTG GTC TGT TCC CTG GAC GGG CTG TTC CCA GTC TCG GAG GCCGln Gly Thr Val Val Cys Ser Leu Asp Gly Leu Phe Pro Val Ser Glu AlaCAG GTC CAC CTG GCA CTG GGG GAC CAG AGG TTG AAC CCC ACA GTC ACC TATGln Val His Leu Ala Leu Gly Asp Gln Arg Leu Asn Pro Thr Va Thr TyrlGGC AAC GAC TCC TTC TCG GCC AAG GCC TCA GTC AGT GTG ACC GCA GAG GACGly Asn Asp Ser Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu AspGAG GGC ACC CAG CGG CTG ACG TGT GCA GTA ATA CTG GGG AAC CAG AGC CAGGlu Gly Thr Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser GlnGAG ACA CTG CAG ACA GTG ACC ATC TAC AGC TTT CCGGlu Thr Leu Gln Thr Val Thr Ile Tyr Ser Phe Pro。
(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 H-ICAM-1's 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: before the treatment after the sick group of Graves ', the pharmacological agent thyroid function recover the sick group of normal Graves ' serum sICAM-1 apparently higher than normal group (P＜0.01), and the big patient's group of simple goiter does not have with normal group and steams different (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 before and after the full excision of Tiroidina time,
131Before and after the I treatment and before and after the antithyroid drug treatment, show 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 (10)
1. the outer I-III function area gene expression product of a people intercellular adhesion molecule-1 film, it is characterized in that this expression product can substitute the nucleotide sequence of people's intercellular adhesion molecule-1 total length, its recombinant protein has immunocompetence, its fusion protein molecule amount is 66.37kD, and productive rate is about the 35mg/L substratum.
2. function area gene expression product according to claim 1 is characterized in that the recombinant plasmid called after ZpUCICAM that extracts, and its gene fragment size is consistent with want clone gene fragment.
3. function area gene expression product according to claim 1 is characterized in that the recombinant plasmid called after ZpET42aICM of function area gene expression vector, and its interface sequence, amino acid single open reading frame are:
CC ATG GGA TAT CGG 
CAG ACA TCT GTG TCC CCThe PET42a carrier
Goal gene.
4. 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, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium; In the affinity purification post, purifying obtains expression product GST-ICAM-1, and is multiple through affinitive layer purification, obtains the outer I-III functional zone fusion rotein GST-ICAM-1 of reorganization ICAM-1 film.
5. the preparation method of expression product according to claim 5 is characterized in that recombinant expressed type plasmid is transformed into E.coli BL21 and expresses in the bacterium and express.
6. the preparation method of expression product according to claim 5, it is characterized in that recombinant expressed type plasmid is transformed into the expression condition that E.coli BL21 expresses in the bacterium and is: with the 0.5-1% lactose is inductor, 20-37 ℃, induces 3-24 hour.
7. the preparation method of expression product according to claim 5 is characterized in that the expression condition that recombinant expressed type plasmid is transformed in the E.coli BL21 expression bacterium is: be inductor with IPTG0.1-1.0mM.
8. the preparation method of expression product according to claim 5 is characterized in that the outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid PET28b, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium.
9. the preparation method of expression product according to claim 5 is characterized in that the outer I-III functional zone of ICAM-1 film encoding gene is recombinated among the expressive plasmid PGEX4T3, more recombinant expressed type plasmid is transformed in the prokaryotic expression bacterium.
10. 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, obtain the anti-people's of rabbit polyclonal antibody behind the immunizing rabbit, again with the anti-people's of rabbit polyclonal antibody as an anti-human serum sICAM-1 radiating immuning analysis technology that is used for.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02103820 CN1243097C (en) | 2002-03-29 | 2002-03-29 | Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02103820 CN1243097C (en) | 2002-03-29 | 2002-03-29 | Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1448509A true CN1448509A (en) | 2003-10-15 |
| CN1243097C CN1243097C (en) | 2006-02-22 |
Family
ID=28680019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02103820 Expired - Fee Related CN1243097C (en) | 2002-03-29 | 2002-03-29 | Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1243097C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936604A (en) * | 2012-11-09 | 2013-02-20 | 四川大学 | Human hcrcn81 pronucleus recombinant plasmid, protein and rabbit anti-human polyclonal antibody expressed by same and application |
| CN108982877A (en) * | 2018-09-07 | 2018-12-11 | 成都创宜生物科技有限公司 | A kind of sICAM-1 fluorescence immune chromatography test strip, preparation method and applications |
-
2002
- 2002-03-29 CN CN 02103820 patent/CN1243097C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936604A (en) * | 2012-11-09 | 2013-02-20 | 四川大学 | Human hcrcn81 pronucleus recombinant plasmid, protein and rabbit anti-human polyclonal antibody expressed by same and application |
| CN108982877A (en) * | 2018-09-07 | 2018-12-11 | 成都创宜生物科技有限公司 | A kind of sICAM-1 fluorescence immune chromatography test strip, preparation method and applications |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1243097C (en) | 2006-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lamkhioued et al. | Human eosinophils express a receptor for secretory component. Role in secretory IgA‐dependent activation | |
| JP4085114B2 (en) | Novel chemokines expressed in inflammatory adenoids and their production and use | |
| EP0613006A1 (en) | Antibodies to cachectin and immunoassays | |
| JPH09118631A (en) | Tumor necrosis factor suppresive protein | |
| US5602008A (en) | DNA encoding a liver expressed chemokine | |
| NZ229514A (en) | Nucleic acid encoding carcinoembryonic antigens (cea) and sequence specific antibodies derived therefrom | |
| KR19980064719A (en) | Interleukin-18-Receptor Protein | |
| US7803373B2 (en) | Chemokine panec-1 antibodies | |
| CA2207009A1 (en) | A new chemokine expressed in fetal spleen, its production and uses | |
| CA2925004A1 (en) | Biomarkers for systemic lupus erythematosus disease activity, and intensity and flare | |
| WO2009025743A2 (en) | Use of trail compositions as antiviral agents | |
| CN113286633A (en) | Bispecific CD123x CD3 diabodies for the treatment of hematological malignancies | |
| US5852176A (en) | Antibodies to receptors for human interleukin-12 | |
| KR19990063764A (en) | C-type chemokine type protein | |
| CN1396183A (en) | Human fusion antibody for reducing cerebral amyloid fibers associated with senile dementia | |
| CN1448509A (en) | Human ICAM-1 extramembranous I-III domain gene expression product ,its prep. and application in radioimmunological technology | |
| US6489138B1 (en) | Human ependymin | |
| CN103073636A (en) | Dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications thereof | |
| KR890700605A (en) | Conventional cancer sympathetic SCM recognition factors, their manufacture and use | |
| AU701311B2 (en) | Thyroxin-binding proteins | |
| CN103059119A (en) | Dust mite allergen Derf28 and genes and application thereof | |
| JPH05509325A (en) | Nuclear phosphoproteins for predicting lymphoid or epithelial cancer | |
| CN1377408A (en) | The PRV-1 gene and use thereof | |
| CN112316117A (en) | Application of recombinant protein hID2 in preparation of colitis treatment drug | |
| JP2000500970A (en) | Chemokines from pancreas of non-insulin dependent diabetic patients |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060222 Termination date: 20160329 |