Liu Ben, Zhong Mintao, Lun Yongzhi, Wang Xiaoli, Sun Wenchang, Li Xingyun, Ning Anhong, Cao Jing, Zhang Wei, Liu Lei, Huang Min
Department of Microbiology, Dalian Medical University, No. 9 West Section, Lvshun South Road, Lvshunkou District, Dalian 116044, China.
Int J Mol Sci. 2012;13(5):6246-6265. doi: 10.3390/ijms13056246. Epub 2012 May 21.
An apoptosis correlated molecule-protein Latcripin-1 of Lentinula edodes C(91-3)-was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C(91-3). According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3'-Full Rapid Amplification of cDNA Ends (RACE) and 5'-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.
香菇C(91 - 3)中的一种凋亡相关分子蛋白Latcripin - 1在毕赤酵母GS115中进行了表达和特性分析。从香菇C(91 - 3)中提取总RNA。根据转录组，采用3'- cDNA末端快速扩增(RACE)和5'- RACE方法分离出Latcripin - 1的全长基因。将全长基因插入分泌表达载体pPIC9K。Latcripin - 1蛋白在毕赤酵母GS115中表达，并通过十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS - PAGE)和蛋白质免疫印迹法进行分析。蛋白质免疫印迹法表明该蛋白成功表达。采用流式细胞术和3-(4,5 - 二甲基噻唑 - 2 - 基)- 2,5 - 二苯基四氮唑溴盐(MTT)法研究了Latcripin - 1蛋白对A549细胞的生物学功能。通过将鉴定后的蛋白与鸡胚成纤维细胞共培养，用MTT法检测Latcripin - 1蛋白的毒性作用。MTT法检测结果显示，Latcripin - 1蛋白组与对照组之间存在显著差异(p < 0.05)。鉴定后的蛋白对鸡胚成纤维细胞无毒性作用。流式细胞术显示，根据凋亡功能，目标蛋白组与对照组之间存在显著差异(p < 0.05)。同时，透射电子显微镜观察到的细胞超微结构支持了流式细胞术的结果。该研究表明，Latcripin - 1蛋白可诱导人肺癌细胞A549凋亡，为寻找抗肿瘤蛋白带来了新的见解和优势。
