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Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP)

A technology for quantitative determination and establishment of methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, cells modified by introducing foreign genetic materials, etc. Cell lines or cytological methods and other issues, to achieve the effect of simple operation and sensitive indicators

Inactive Publication Date: 2002-07-10
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

[0003] The existing bone morphogenetic protein (BMP) activity assay method, that is, the animal implantation experiment, has the disadvantages of long cycle, inability to quantify, and being affected by various factors such as animal species and implantation site.
So far there is no cell line or cytological method specifically for quantitative determination of bone morphogenetic protein

Method used

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  • Establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP)

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Embodiment Construction

[0010] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. By the method of the present invention:

[0011] a. First synthesize the osteocalcin partial promoter (6OCP) with 6 key elements of BMP in series, the 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', connected to the luciferase reporter gene, cloned and constructed its eukaryotic expression vector, so that the luciferase reporter gene is under the control of the promoter (6OCP);

[0012] b. Then transfer into myoblast C2C12, use the resistance gene of the carrier itself to carry out pressure screening with antibiotics, and obtain a monoclonal cell line that can be induced by BMP and stably express the luciferase reporter gene;

[0013] The above-mentioned eukaryotic expression vectors can also be transferred into other cell lines, such as NIH3T3, ...

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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

1. Technical field: [0001] The invention relates to a method for detecting cytokine activity in biotechnology, and further relates to a method for establishing a cell line for quantitative determination of bone morphogenic protein (BMP) activity. 2. Background technology [0002] The existing bone morphogenetic protein (BMP) activity assay method is mainly the classic animal implantation experiment, the specific method is: surgically cut the skin or deep muscle of the experimental animal, put a certain amount of BMP into it, and suture the wound ; Then at different times after the operation, generally 2 to 4 weeks, samples were taken from the implantation site, histological sections were made, stained, and finally the osteoinductive activity of BMP was observed under a microscope. [0003] The existing bone morphogenetic protein (BMP) activity assay method, that is, the animal implantation experiment, has the disadvantages of long cycle, inability to quantify, and being affe...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/06C12Q1/25
Inventor 朱帮福陈苏民陈南春
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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