大连医科大学学报
大連醫科大學學報
대련의과대학학보
JOURNAL OF DALIAN MEDICAL UNIVERSITY
2015年
3期
214-219
,共6页
曹淑云%刘奔%钟民涛%伦永志%张伟%王晓丽%李星云%曹靖%宁安红%黄敏
曹淑雲%劉奔%鐘民濤%倫永誌%張偉%王曉麗%李星雲%曹靖%寧安紅%黃敏
조숙운%류분%종민도%륜영지%장위%왕효려%리성운%조정%저안홍%황민
香菇C91-3%克隆%重组蛋白
香菇C91-3%剋隆%重組蛋白
향고C91-3%극륭%중조단백
Lentinula edodes C91-3%clone%recombinant protein
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pET32a（＋）－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。
目的：剋隆香菇C91－3轉錄組中Unigene 28447基因，對其進行生物信息學分析，併研究該基因結構域的生物學活性。方法提取香菇C91－3菌絲體中的總RNA，用3′－Full RACE、5′－Full RACE方法穫得香菇菌絲體Unigene 28447基因全長；構建基因錶達載體，進行原覈誘導錶達，將錶達蛋白進行純化、複性。運用MTT法、流式細胞儀法研究蛋白對腫瘤細胞生長、週期及凋亡的影響。結果成功構建重組質粒pET32a（＋）－Unigene，誘導重組蛋白錶達，併進行純化、複性。 MTT法顯示不同濃度蛋白對腫瘤細胞的生長抑製作用與對照組比較差異均有顯著性意義（P＜0．05），流式細胞儀顯示此蛋白具有誘導腫瘤細胞改變週期和凋亡的作用（P＜0．05）。結論初步鑒定Unigene 28447基因的結構域具有抗腫瘤活性。
목적：극륭향고C91－3전록조중Unigene 28447기인，대기진행생물신식학분석，병연구해기인결구역적생물학활성。방법제취향고C91－3균사체중적총RNA，용3′－Full RACE、5′－Full RACE방법획득향고균사체Unigene 28447기인전장；구건기인표체재체，진행원핵유도표체，장표체단백진행순화、복성。운용MTT법、류식세포의법연구단백대종류세포생장、주기급조망적영향。결과성공구건중조질립pET32a（＋）－Unigene，유도중조단백표체，병진행순화、복성。 MTT법현시불동농도단백대종류세포적생장억제작용여대조조비교차이균유현저성의의（P＜0．05），류식세포의현시차단백구유유도종류세포개변주기화조망적작용（P＜0．05）。결론초보감정Unigene 28447기인적결구역구유항종류활성。
Objective To clone and express the Lentinula edodes C91-3 transcriptome Unigene 28447 and to investigate its biological activity.Methods Lentinula edodes C91-3 mycelium total RNA were extracted.The full-length gene of Lentinu-la 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 pET32a( +).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 anti-tumor activity was found in the protein, which would be the basis for further study in the future.
