Application of GCA stable transfection cell line to test BNP (Brain Natriuretic Peptide) activity

A transgenic cell and cell technology, applied in the measurement/inspection of microorganisms, cells modified by introducing foreign genetic material, biochemical equipment and methods, etc., can solve the problems of large errors and cumbersome operations, and achieve small variation and high application Value, objective and reliable results

Active Publication Date: 2013-10-30
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is not only cumbersome to operate, but also has relatively large errors.

Method used

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  • Application of GCA stable transfection cell line to test BNP (Brain Natriuretic Peptide) activity
  • Application of GCA stable transfection cell line to test BNP (Brain Natriuretic Peptide) activity
  • Application of GCA stable transfection cell line to test BNP (Brain Natriuretic Peptide) activity

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Experimental program
Comparison scheme
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Embodiment Construction

[0043] 1. Materials and Methods:

[0044] 1.1 Research object: B-type brain natriuretic peptide (BNP)

[0045] 1.2 NPR1 / GCA plasmid: the gene ID is NM_000906, purchased from Aurui Dongyuan Gene Co., Ltd., see the vector map image 3 .

[0046] HEK293 cells: stored in the cell room of China Food and Drug Research Institute.

[0047] BNP reference product: prepared by the Recombinant Technology Product Office of China Institute of Food and Drug Research and Testing.

[0048] 1.3 Reagents:

[0049] Growth medium: DMEM (GIBCO, #11995) + 10% fetal bovine serum (GIBCO, #10099) + 1% double antibody (GIBCO, #15240)

[0050] Plate medium: DMEM (GIBCO, #11995)

[0051] Liposome 2000: Invitrogen, #11668

[0052] G418: CalBiochem, #345810

[0053] G418 selection medium: growth medium + 200ug / ml G418

[0054] BNP diluent: PBS+0.2%BSA (Merck Calbiochem, #12659)+1mM IBMX (sigma, I5879, add before use)

[0055] cGMP detection kit: cell signaling, #4360S

[0056] cGMP primary antib...

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Abstract

The invention discloses a method for testing BNP (Brain Natriuretic Peptide) activity by using a GCA stable transfection cell line and establishes a novel BNP bioactivity testing method. GCA which is a main acceptor of BNP is an adenylate cyclase precursor and can catalyze GTP (guanosine triphosphate) to generate cGMP after being activated by the BNP and then exerts a biological effect. Wield 293 cell has low GCA expression level and low reactivity to BNP. In the method disclosed by the invention, a stable cell line 293GCAC3 with BNP acceptor (GCA) overexpression level is structured by transgenosis, the content change of cGMP secreted into cell supernatant by the 293GCAC3 stimulated by the BNP is detected to quantify the activity of the BNP. The method comprises the following specific testing steps of: laying cells on a 96-pore plate and culturing for 16-18 hours; adding the BNP for stimulating for 90 minutes; and sucking cell supernatant and detecting the content of cGMP with competitive ELISA (enzyme-linked immuno sorbent assay). The method is simple to operate, has sensitive response and small variability and is significant for the quality control and clinical application of the BNP.

Description

Technical field: [0001] The invention relates to the field of biopharmaceutical activity detection, and establishes a faster and more accurate quantitative method for the activity measurement of brain natriuretic peptide (BNP). Background technique: [0002] The current method for measuring the activity of BNP is the isolated arterial strip method, and its activity is measured by recording the change of arterial tension under the action of BNP. This method is not only cumbersome to operate, but also has relatively large errors. The in vitro activity assay method based on cell lines is easy to operate and has small errors, and is being more and more widely used in the activity detection of biological drugs. Therefore, we hoped that a cell line-based activity assay could be established. Considering that there is no suitable cell line to measure the activity of BNP, we first constructed a stable cell line with overexpression of BNP receptor (GCA) through transgene, and then e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12N5/10
Inventor 王军志饶春明于雷高凯史新昌
Owner NAT INST FOR FOOD & DRUG CONTROL
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