香菇C_(91-3)转录本Unigene14872基因Pkinase结构域的克隆表达及其生物信息学分析

Cloning and Bioinformatics Analysis of Pkinase Structure Domain in Unigene 14872 Gene from Xianggu Mushroom (Lentinula edodes) C_(91-3) Transcript

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.