天麻免疫調節蛋白之純化與活性分析

Abstract

Tianma (Gastrodia elata), also known as fixed wind grass or barbed arrows, is a medicinal orchid used in traditional Chinese medicine to treat vertigo, paralysis, convulsions, cramps, epilepsy, high blood pressure and other symptoms. We have previously purified an immunomodulatory protein IPAF from Anoectochilus formosanus. The protein IPAF has an amino acid sequence highly similar to proteins from Orchidaceae plants, including Epipactis helleborine lectin, Listera ovata lectin, and G. elata-gastrodianin-4B. Therefore, we hypothesized that Orchidaceae plants might possess proteins similar to IPAF. To test our hypothesis, the crude protein was extracted from G. elata, Dendrobium huoshanense, Dendrobium nobile, and Dendrobium loddigesii and then analyzed by polyacrylamide gel electrophoresis and Western blotting. Subsequently, a 15.1 kDa protein G. elata (GEP), which exhibited similar biochemical characteristics to IPAF, was purified via anion exchange chromatography using DE-52 and HiTrap Q columns. Finally, RAW 264.7 murine macrophage cells were used for preliminary activity tests. GEP (5 μg/mL) could activate nitric oxide (NO) (10.4 ± 0.6 μM), TNF-α (935.4 ± 118.5 pg/mL), and IL-1β (25.1 ± 3.0 pg/mL) secretion by RAW 264.7 murine macrophage. In addition, GEP enhanced nucleic acid replication by 1.16-fold as analyzed by BrdU analysis, and promoted 1.43-fold of MTT metabolic activity in murine splenocytes. In this study we purified a new orchid protein, which exhibited similar biochemical characteristics as the IPAF, and we also assessed its immunomodulatory function. GEP activated RAW 264.7 macrophages by enhancing NO, TNF-α and IL-1β secretion. Moreover, GEP also stimulated cell proliferation in mouse splenocytes. On the basis of previous the findings, in our laboratory GEP, as well as other orchid proteins, might play important roles in the medicinal functions of Orchidaceae plants.