香菇C_(91-3)转录组Unigene 28447基因的蛋白表达及其对细胞凋亡和周期的影响

Expression of recombination Unigene 28447 of Lentinula edodes C_(91-3) and its effect on apoptosis and cell cycle of lung cancer cell A549

目的克隆香菇C91-3转录组中Unigene 28447基因,对其进行生物信息学分析,并研究该基因结构域的生物学活性。方法提取香菇C91-3菌丝体中的总RNA,用3'-Full RACE、5'-Full RACE方法获得香菇菌丝体Unigene 28447基因全长;构建基因表达载体,进行原核诱导表达,将表达蛋白进行纯化、复性。运用MTT法、流式细胞仪法研究蛋白对肿瘤细胞生长、周期及凋亡的影响。结果成功构建重组质粒p ET32a(+)-Unigene,诱导重组蛋白表达,并进行纯化、复性。MTT法显示不同浓度蛋白对肿瘤细胞的生长抑制作用与对照组比较差异均有显著性意义(P<0.05),流式细胞仪显示此蛋白具有诱导肿瘤细胞改变周期和凋亡的作用(P<0.05)。结论初步鉴定Unigene 28447基因的结构域具有抗肿瘤活性。

Objective To clone and express the Lentinula edodes C91- 3transcriptome Unigene 28447 and to investigate its biological activity. Methods Lentinula edodes C91- 3mycelium total RNA were extracted. The full- length gene of Lentinula edodes mycelium Unigene 28447 were obtained with 3＇- Full RACE,5＇- Full RACE kits. The gene expressed in E. coli after induction,then the protein was extracted,purified and refolded. MTT assay and flow cytometry were used to study the tumor cell growth,apoptosis and cell cycle. Results The recombinant protein was successfully induced and expressed in plasmid p ET32a（ ＋）. MTT assay showed that different concentrations of this protein inhibited A549 growth significantly as compared with control group（ P〈0. 05）. Flow cytometry showed this protein induced apoptosis of A549 and changed cell cycle significantly（ P〈0. 05）. Conclusion The antitumor activity was found in the protein,which would be the basis for further study in the future.