RT-PCR-based cloning of the human beta-amyloid precursor protein gene and the construction of its expression plasmids

摘要

Beta-amyloid peptide (betaA) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. betaA generation depends on proteolytic cleavage of the amyloid precursor protein (APP). The present invention is a new procedure for the cloning of human betaA precursor protein gene (human APP gene) based on the reverse transcription (RT) and the polymerase chain reaction (PCR). This procedure for cloning human APP gene by means of RT-PCR reactions is cost-effective, not time-consuming, and is suited for any laboratory. The present invention also includes a new procedure for the construction of expression plasmids, a/ using the pFastBac(TM) HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human APP in insect cells; and b/ using the pET-28a (+) transfer vector for the purpose of obtaining human APP in bacteria. The present invention makes it easier to obtain full-length APP which is essential for the identification of biological activities that occur in the APP molecule and for the identification of proteases capable of creating betaA. Knowing which protease creates betaA is important for the exploration of therapeutic and preventative strategies for the treatment of Alzheimer's disease.