Abstract: Objective To develope a novel BMP-2 bioactivity detection system based on a dual-luciferase reporter gene stably transfected cell line (C2C12^BBDE) to address the issues of low sensitivity, time-consuming procedures in traditional alkaline phosphatase ( ALP) activity assays, and cumbersome operational workflows in real-time fluorescent quantitative polymerase chain reaction(qRT-PCR ) methods. Methods This system integrates a BMP-2 responsive promoter driving NanoLuc luciferase (NLuc) expression with constitutively expressed Firefly luciferase ( Fluc) as an internal control via lentiviral transduction. Potential promoter interference was mitigated through strategic design of an inverse transcription unit and PolyA sequence insertion. Results This novel system achieved a dynamic detection range of 0-30 ng / mL, matching the sensitivity of qRT-PCR while demonstrating a statistically significant improvement over the ALP assay. Furthermore, the assay cycle was markedly reduced to 16 hours, representing an efficiency enhancement exceeding 700 % relative to the ALP method. Critically, the workflow is streamlined, obviating the need for complex RNA extraction and reverse transcription steps inherent in qRT-PCR. Conclusion The successful establishment of this system not only furnishes a standardized, high-throughput tool for BMP-2 bioactivity assessment but also provides a modular, adaptable technical paradigm for the development of bioactivity assays for other growth factors. This advancement holds substantial translational potential, significantly contributing to the rational development and clinical implementation of bone regenerative therapeutics.

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