香菇C_(91-3)转录本Unigene8290基因结构域片段的克隆表达及其抗肿瘤活性初步研究  

The cloning,expression and biological activity of Unigene8290 transcript fromLentinula edodes C_(91-3)

作　　者：郭雪芳[1] 阮文静[1] 王晓丽[1] 沙丽[1] 李星云[1] 刘奔[1] 张伟[1] 高一凡[1] 曹婧[1] 宁安红[1] 黄敏[1] 钟民涛[1] 

机构地区：[1]大连医科大学微生物教研室,辽宁大连116044

出　　处：《中国微生态学杂志》2016年第5期501-505,共5页Chinese Journal of Microecology

基　　金：国家自然科学基金项目(81301995)

摘　　要：目的通过对香菇C91-3转录组进行筛选,并克隆表达含RCC1(染色体浓缩调控蛋白)和ANK(锚蛋白)结构域的Unigene8290基因,研究其抗肿瘤活性。方法从香菇C91-3菌丝体中提取总RNA,反转录合成cDNA,并利用Rapid Amplification of cDNA Ends(RACE)技术获得基因全长。生物信息学分析其结构域。设计特有引物,采用PCR技术扩增该结构域,将其克隆产物与pET-32a(+)载体连接,热转化至E.coil Rosetta-gami(DE3)中诱导表达,纯化、复性后,通过MTT法研究其抗肿瘤活性。结果生物信息学显示Unigene8290基因具有RCC1和ANK结构域,琼脂糖凝胶电泳与测序结果显示Unigene8290基因的结构域片段与pET-32a(+)载体重组成功,SDS-PAGE与Western blot结果显示Unigene8290蛋白成功表达,MTT结果显示该重组融合蛋白对HepG2细胞生长具有明显的抑制作用,并且其抑制作用存在一定的时间和浓度依赖性。结论成功诱导Unigene8290的RCC1和ANK结构域蛋白表达,并初步鉴定其具有抑制HepG2肿瘤细胞增殖活性的功能。Objective To clone and express the Unigene8290 gene containing ANK and RCC1 domains of Lentinula edodes C91-3transcripts,and study its anti-tumor activity.Methods Total RNAs were extracted from the mycelia of Lentinula edodes C91-3,then cDNA was synthesized by reverse transcription,and the full-length gene was obtained by Rapid Amplification of cDNA Ends（RACE）technology;bioinformatics method was used to analyze its domains.The RCC1 and ANK domains were amplified by PCR,and the cloned products were connected with pET-32a（＋）vector.The recombined vector was transformed into Rosetta-gami（DE3）and induced,purified and refolded.MTT method was used to detect its anti-tumor activity.Results Bioinformatics analysis showed that the Unigene8290 had RCC1and ANK domains.Agarose gel electrophoresis showed that the ANK and RCC1 domains of Unigene8290 were constructed with pET-32a（＋）vector successfully.SDS-PAGE and Western blot results showed that the protein Unigene8290 was expressed successfully.MTT results showed that the protein had an inhibitory effect on HepG2 cells obviously,in a time-and concentration-dependent way.Conclusion The Unigene8290 was cloned and its expression induced successfully,which can inhibit the proliferation of tumor cells.

关 键 词：香菇蛋白 克隆 生物信息学 结构域 

分 类 号：Q943[生物学—植物学]