摘要
旨在克隆香菇C91-3转录本Unigene 14872基因中的Pkinase结构域,并对其进行生物信息学分析。提取香菇C91-3菌丝体中总RNA,根据转录组测序结果,用3'-Full RACE、5'-Full RACE方法获得Unigene 14872基因全长。用NCBI对其进行生物信息学分析,预测其具有丝氨酸/苏氨酸蛋白激酶(Pkinase)结构域。设计引物PCR扩增Pkinase结构域,然后克隆至pET32a(+)载体,再热转化至JM109中保存并进行质粒测序。重组质粒pET32a(+)-Pkinase构建成功,并成功诱导表达重组蛋白,初步鉴定该蛋白具有抗肿瘤活性,为进一步研究重组蛋白的活性及作用机制奠定基础。
The aim of this study was to clone the Pkinase structure domain in Unigene 14872 gene from Xianggu mushroom (Lentinula edodes) C91-3 transcription, and carried out bioinformatics analysis. Total RNA from hyphae of L. edodes C91-3 was extracted; then the full length gene of Unigene 14872 was gotten with 3'-Full RACE and 5'-Full RACE method according to the transcription group sequencing results. And carried out bioinformatics analysis with NCBI, and forecasted Pkinase possessing serine/threonine protein kinase (Pkinase) structure domain. And design a primer to PCR amplify Pkinase structure domain, and then cloned into pET32a ( + ) vector, and heat-transformed into JM109 to store and carry out plasmids sequencing. The recombinant plasmid pET32a ( + )-Pkinase was successfully constructed, and successfully induced the expression of recombinant protein. It was initially identified the protein possessed anti-tumor activity. These laid a foundation for further study on the activity and mechanism of the recombinant protein.
出处
《微生物学杂志》
CAS
CSCD
2014年第1期22-27,共6页
Journal of Microbiology
基金
国家自然科学基金(30770018)
关键词
香菇C91-3
克隆
生物信息学
蛋白激酶
Xianggu mushroom ( Lentinula edodes) C91-3
clone
bioinformaties
protein kinase