CN1357621A - Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP) - Google Patents
Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP) Download PDFInfo
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- CN1357621A CN1357621A CN 01131813 CN01131813A CN1357621A CN 1357621 A CN1357621 A CN 1357621A CN 01131813 CN01131813 CN 01131813 CN 01131813 A CN01131813 A CN 01131813A CN 1357621 A CN1357621 A CN 1357621A
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- bmp
- cell strain
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- ccaac
- luciferase
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Abstract
The present invention relates to establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP). By means of the transgenic technology of molecular biology, partial promoter of BMP action target molecular osteocalcium together with leuciferinase report gene is transfered to myogenic cell C2C12, monoclonal cell strain to express leuciferinase report gene stably is obtained through screening and the reaction of the cell strains to BMP action is determined. Strain with high reaction strength to BMP stimulation and sensitive determination index is finally sorted. Specificity and stability test proves the stability and reliability of the cell strain and multiple BMP sample testing result shows that the action of BMP is correlated with leuciferinase activity linearly and directly. The cell strain of the present invention is suitable for quantitative determination of BMP activity.
Description
One, technical field:
The present invention relates to the detection method of the cytokine activity in a kind of biotechnology, further relate to the establishment method of a kind of quantitative assay bone morphogenetic protein (BMP) active cell strain.
Two, background technology
Existing bone morphogenetic protein (BMP) activity determination method is mainly classical animal body and is implanted into experiment, and concrete grammar is: the skin of surgical incision laboratory animal or be deep to muscle, and a certain amount of BMP is put into wherein, and sew up a wound; In the postoperative different time, be generally for 2～4 weeks then, draw materials, do Histological section, dye, examine under a microscope the bone-inducing activity of BMP at last at implant site.
Existing bone morphogenetic protein (BMP) activity determination method, promptly animal body is implanted into experiment, exist the cycle long, can not be quantitative, and be subjected to the shortcoming of multiple factor affecting such as animal varieties, implant site.So far still there are not the cell strain or the cytology method that are specifically designed to the quantitative assay bone morphogenetic protein.
Three, summary of the invention
According to defective or the deficiency that above-mentioned prior art partly exists, the purpose of this invention is to provide a kind of establishment method that is specifically designed to quantitative assay bone morphogenetic protein (BMP) active cell strain.
Bone Gla protein is a kind of essential stromatin of scleroblast excretory in the bone forming process, and it is one of the important target molecule of bone morphogenetic protein (BMP) effect just.
The technical solution used in the present invention is: the Bone Gla protein part promotor (6OCP) of 6 BMP effect key elements of at first synthetic series connection, (sequence is: luciferase reporter gene in the connection 5 '-CCAAC CACA CCAAC CACA CCAACCACA CCAAC CACA CCAAC CACA CCAAC CACA GCC GAT ATA AAT GCTACT GGA TGC TGG AGG GTG CAG AAC AGA CAA GTC-3 '), clone, make up its carrier for expression of eukaryon, luciferase reporter gene is under this promotor (6OCP) control.Then above-mentioned carrier is changed among the sarcoplast C2C12, the resistant gene that utilizes expression vector itself carries out the pressure screening with microbiotic, and acquisition can be subjected to that BMP induces, the monoclonal cell strain of stably express luciferase reporter gene.The expression product of luciferase reporter gene is a luciferase, and the activity of luciferase can record by detecting its bottom exploded deposits yields fluorescence volume.Concrete detection method is: collect the monoclonal cell that BMP stimulates, cultivates 2 days, add cell pyrolysis liquid, treated after the complete cracking of cell high speed centrifugation 5 minutes, get the centrifugal supernatant of part then, join in a certain amount of luciferase effect substrate solution, on chemiluminescence detector, measure and produce fluorescence volume.By detecting the reactivity of each monoclonal cell strain to the BMP effect; Finally select BMP irritant reaction intensity height, detect index (luciferase generation fluorescence volume) sensitive cell strain.
Method of the present invention verifies through specificity, stability etc., and this cell strain is reliable and stable, is applicable to the active quantitative assay of BMP.
The activity of utilizing the present invention to measure BMP only needs 2 day time, and easy and simple to handle, and the index sensitivity of surveying can be carried out quantitative analysis.
Four, description of drawings
Fig. 1 is a synoptic diagram of the present invention.
Five, embodiment
Below in conjunction with drawings and Examples the present invention is described in further detail.Press method of the present invention:
The Bone Gla protein part promotor (6OCP) of a. at first synthesizing 6 BMP effect key elements of series connection, sequence is: 5 '-CCAAC CACA CCAAC CACA CCAAC CACA CCAAC CACACCAAC CACA CCAAC CACA GCC GAT ATA AAT GCT ACT GGA TGC TGGAGG GTG CAG AAC AGA CAA GTC-3 ', luciferase reporter gene in the connection, clone, make up its carrier for expression of eukaryon, luciferase reporter gene is under this promotor (6OCP) control;
B. change over to then among the sarcoplast C2C12, the resistant gene that utilizes carrier itself carries out the pressure screening with microbiotic, and acquisition can be subjected to that BMP induces, the monoclonal cell strain of stably express luciferase reporter gene;
Above-mentioned carrier for expression of eukaryon also can change other clone over to, in NIH3T3, C3H10T1/2 cell;
C. by detecting the reactivity of each cell strain to the BMP effect:
Concrete detection method is: collect the monoclonal cell that BMP stimulates, cultivates 2 days, add cell pyrolysis liquid, treated after the complete cracking of cell high speed centrifugation 5 minutes, get the centrifugal supernatant of part then, join in a certain amount of luciferase effect substrate solution, on chemiluminescence detector, measure and produce fluorescence volume;
D. finally select BMP irritant reaction intensity height, detect index sensitive cell strain;
E. verify through specificity, stability etc., this cell strain is reliable and stable, is applicable to the active quantitative assay of BMP;
After the BMP of employing different sources, different batches stimulates this cell strain, detect uciferase activity, the result shows that the effect dosage of BMP and uciferase activity are linear positive correlation.
Claims (1)
1. the establishment method of quantitative assay bone morphogenetic protein (BMP) active cell strain may further comprise the steps:
A. the Bone Gla protein part promotor of 6 BMP effect key elements of at first will connecting connect to go up luciferase reporter gene, clones, makes up its carrier for expression of eukaryon; This promoter sequence is: 5 '-CCAAC CACACCAAC CACA CCAAC CACA CCAAC CACA CCAAC CACA CCAAC CACAGCC GAT ATA AAT GCT ACT GGA TGC TGG AGG GTG CAG AAC AGA CAAGTC-3 ';
B. change over to then among the sarcoplast C2C12, utilize the resistant gene of carrier itself to carry out the pressure screening, obtain the monoclonal cell strain of stably express luciferase reporter gene with microbiotic; The expression product of luciferase reporter gene is a luciferase, and the activity of luciferase can record by detecting its bottom exploded deposits yields fluorescence volume;
Above-mentioned carrier for expression of eukaryon also can change other clone over to, in NIH3T3, C3H10T1/2 cell;
Concrete detection method is: collect the monoclonal cell that BMP stimulates, cultivates 2 days, add cell pyrolysis liquid, treated after the complete cracking of cell high speed centrifugation 5 minutes, get the centrifugal supernatant of part then, join in a certain amount of luciferase effect substrate solution, on chemiluminescence detector, measure and produce fluorescence volume;
C. by detecting the reactivity of each cell strain, finally select BMP irritant reaction intensity height, detect index-luciferase decomposition generation fluorescence volume sensitive cell strain the BMP effect;
D. verify through specificity, stability etc., this cell strain is reliable and stable, is applicable to the active quantitative assay of BMP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011318139A CN1139654C (en) | 2001-12-07 | 2001-12-07 | Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011318139A CN1139654C (en) | 2001-12-07 | 2001-12-07 | Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1357621A true CN1357621A (en) | 2002-07-10 |
| CN1139654C CN1139654C (en) | 2004-02-25 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB011318139A Expired - Fee Related CN1139654C (en) | 2001-12-07 | 2001-12-07 | Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355889C (en) * | 2004-10-11 | 2007-12-19 | 中国人民解放军第四军医大学 | Design constructing method of gene engineering recombined dyad bone shape protein |
| CN1784496B (en) * | 2003-05-06 | 2010-08-25 | 独立行政法人产业技术综合研究所 | Multiple gene transcription activity determining system |
-
2001
- 2001-12-07 CN CNB011318139A patent/CN1139654C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1784496B (en) * | 2003-05-06 | 2010-08-25 | 独立行政法人产业技术综合研究所 | Multiple gene transcription activity determining system |
| CN100355889C (en) * | 2004-10-11 | 2007-12-19 | 中国人民解放军第四军医大学 | Design constructing method of gene engineering recombined dyad bone shape protein |
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| Publication number | Publication date |
|---|---|
| CN1139654C (en) | 2004-02-25 |
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