关键词: 帕金森氏病, α-突触核蛋白, 原纤维, PGEX-5X-1载体, GST

Abstract: The DNA sequence of human α-synuclein was amplified from pcDNA3.1-α-syn vector by PCR, then cloned into PGEX-5X-1 vector, and a highly efficient prokaryotic expression vector of human α-synuclein was successfully constructed and named pGEX-5X-1-syn. The recombinant vector was transformed into BL21 strain and induced by IPTG. The recombinant human α-synuclein fibrils were obtained by optimized the inducing concentration, time and temperature of IPTG, purification method and fibril culture conditions. The preparation of fibrils was confirmed by thioflavin T staining and transmission electron microscopy. Results showed that after inducing by IPTG at a final concentration of 1.0 mmol/L for 3 h at 37 °C, human α-synuclein was expressed in large amounts. The GST fusion protein purification magnetic beads were used for protein purification, and the high-purity human α-synuclein could be obtained quickly and efficiently, and the purified yield reached 25 mg/L. Human α-synuclein fibrils were successfully prepared in large quantities when it was cultured for 6 days at 37 °C at the concentration of 9 mg/mL, shaking at 1000 r/min. The morphology of fibrils was successfully observed under transmission electron microscopy. α-synuclein fibrils are important materials for the establishment of PD models. The above results showed that a method for the rapid and efficient preparation of recombinant human α-synuclein fibrils was established， which provided important research materials for the establishment of animal models and cell models of PD.

Key words: Parkinson′s disease, α-synuclein, fibrils, PGEX-5X-1 vector, GST