水稻OsV3IP2和OsV3IP3原核表达与鉴定

摘要

通过生物信息学分析确定OsV3IP2和OsV3IP3的可溶性肽段,获得可溶性的OsV3IP2和OsV3IP3外源表达蛋白质,将相应的编码区克隆于原核表达载体pET32a中,进行IPTG诱导表达,利用SDS-PAGE和Western blot检测目的蛋白质.结果表明,利用生物信息学软件对蛋白质的二级结构进行预测,将OsV3IP2、OsV3IP3分别分为2个和4个部分.将其分别克隆到pET32a后,采用0.2 mol/L IPTG在10、20、30℃诱导表达.经SDS-PAGE和Western Blot分析,证明OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1表达的融合蛋白存在于上清液中,而OsV3IP 3-2、OsV3IP 3-3表达的融合蛋白存在于沉淀中,获得了OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1可溶性目的蛋白,为进一步体外验证OsVDAC3与OsV3IP2、OsV3IP3的互作奠定了基础.%The bioinformatic analysis was used to identify soluble fragments of OsV3IP2 and OsV3IP3,and soluble OsV3IP2 and OsV3IP3 proteins in bacterial expression systemo were btained.The corresponding coding sequences of different fragments were cloned into bacterial expression vector pET32a.The target peptides were expressed via IPTG induction,and detected by SDS-PAGE and Western blot.Result showed that from the predicted secondary structures,we identified 2 soluble fragments from OsV3IP2 and 4 soluble fragments from OsV3IP3.The expression of recombinant peptides in bacteria was induced in the presence of 0.2 mol/L IPTG at 10,20,or 30 ℃.As shown by SDS-PAGE and Western Blot analysis,OsV3IP2-1,OsV3IP2-2,and OsV3IP3-1 are in the soluble fraction of bacterial lysate,while OsV3IP3-2 and OsV3IP3-3 stay in the precipitation.We have obtained soluble peptides OsV3IP2-1,OsV3IP2-2,and OsV3IP3-1,which laid the foundation for future in vitro studies on protein-protein interactions between OsVDAC3 and OsV3IP2 or OsV3IP3.