蛹蟲草毒性蛋白之生化特性與毒殺作用

Biochemical characteristics and cytotoxicity of the CMP18 protein from Cordyceps militaris

白可鈞(Ke-Chun Bai)

指導教授 ： 許 輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.03331

摘要

蛹蟲草 (Cordyceps militaris) 為蟲草屬研究之模式真菌，乃東方傳統藥材冬蟲夏草之近親。本研究由蛹蟲草純化出一分子量大小約為18 KDa之毒性蛋白Cordyceps militaris protein (CMP18)。蛹蟲草乾燥子實體經過均質、超音波破碎、硫酸銨沉澱、透析後，再依序以CM-52、Resource S陽離子交換管柱層析純化CMP18蛋白。以SDS-PAGE測得其分子量約為18 KDa，由Native-PAGE結果與管柱分離時使用之條件，推測其pI可能大於8.8。CMP18 N端胺基酸序列為GPSVVVGYRTVSAAQAK。醣蛋白染色結果發現CMP18非為醣蛋白，血液凝集試驗顯示蛹蟲草蛋白CMP18對BALB/c小鼠紅血球無凝集作用。
MTT細胞活力試驗得知蛹蟲草蛋白CMP18對於小鼠初代脾臟細胞、小鼠初代腹腔巨噬細胞、小鼠肝癌細胞株BNL (BNL 1MEA.7R.1, ATCC TIB-75)、小鼠白血病巨噬細胞株RAW264.7 (ATCC TIB-71)、小鼠白血病B細胞株X63 (80 V 5B4, TIB-132) 之cell viability皆有抑制作用。後續以小鼠肝癌細胞株BNL為模式進一步探討，發現CMP18蛋白對BNL細胞之活力抑制作用具有時間與劑量上的效應。透過trypan blue細胞染色及測定細胞之乳酸脫氫酶釋放，發現CMP18使生長中的BNL細胞數量減少，並確實有死亡的情形。細胞週期分析指出CMP18蛋白共培養之BNL細胞週期趨向於sub-G1位向，進一步以膜聯蛋白染色與PI雙染分析，顯示CMP18蛋白可能誘導BNL細胞產生細胞凋亡。BNL細胞與CMP18共培養後，有caspase-9活化及粒線體膜電位喪失之情形，推測CMP18可能透過粒線體依賴路徑引發細胞凋亡。蛹蟲草蛋白CMP18經過加熱或強鹼處理，有降解之情形，且失去誘導細胞凋亡之效果；熱或鹼處理可降低CMP18毒性，使蛹蟲草食用上更添安全。

關鍵字

蛹蟲草 ； 蛋白質純化 ； 生化特性 ； 毒蛋白 ； 細胞凋亡

並列摘要

Cordyceps militaris is a model species for Cordyceps research, and is phylogenetically close to an oriental traditional medicine, Cordyceps sinensis. We have purified a new protein from the fruiting body of C. militaris by a series of process such as homogenization, ultra sonication, ammonium sulfate precipitation, and cation exchange chromatography through CM-52 and Resource S columns. The molecular weight of the protein was ~18 kDa as determined by SDS-PAGE analysis and designated as Cordyceps militaris protein 18, CMP18. The results of native-PAGE and the conditions used in column chromatography suggested that the isoelectric point of CMP18 might be greater than 8.8. The N-terminal amino acid sequence determined by automated Edman degradation was GPSVVVGYRTVSAAQAK. The results of PAS staining and hemagglutination assay indicated that CMP18 was not a glycoprotein and did not possess agglutination activity toward mouse red blood cells.
According to MTT cell viability test, CMP18 inhibited the cell viability in mice primary splenocytes and peritoneal macrophages, murine hepatoma cell line BNL, murine leukemia macrophage cell line RAW264.7, and murine leukemia B cell line X63. Further tests on murine hepatoma cell lines BNL revealed that CMP18 inhibited the cell viability in a time- and dose-dependent manner. By trypan blue staining and cell LDH releasing assay, we proved that CMP18 could cause cell death. Furthermore, cell cycle analysis as well as annexin V and PI double stain indicated that CMP18 might mediate BNL cell apoptosis, but not necrosis. Cell caspase-9 was activated and the mitochondria potential was lost after co-incubation with CMP18. These results showed that CMP18 might induce cell apoptosis through a mitochondria dependent pathway. After heat treatment or alkalization, CMP18 would be degraded and lost the ability to induce cell apoptosis, which allowing safe consumtion of Cordyceps militaris.

並列關鍵字

Cordyceps militaris ； protein purification ； biochemical characteristics ； cytotoxicity protein ； apoptosis

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