摘要
为研究芒果MiRab11蛋白的互作及其它生物学功能,本文在MiRab11基因序列基础上,采用重叠PCR定点突变技术,获得了2个结构域突变体Mi-Rab11CA和Mi-Rab11DN,将其构建原核表达载体,并成功转化大肠杆菌BL21。SDS-PAGE结果显示,在28℃条件下,用0.5 mmol/L IPTG诱导4 h,可获得大量可溶性蛋白的表达。将可溶性蛋白经Ni-NTA argrose层析柱纯化并western blot鉴定,该重组蛋白均可与抗His单克隆抗体发生特异性免疫反应。本研究为深入研究芒果pET-Rab11蛋白生理活性、特异性结合位点及功能调控打下良好基础。
In order to study the interaction of MiRab11 with proteins and other biological functions overlapping PCR was applied to obtain two mutant （MiRab11CA and MiRab11DN） based on the known sequence of MiRab11.And the prokaryotic expression vectors of pET-Rab11,pET-Rab11CA,and pET-Rab11DN were successfully constructed and transformed into E.coli BL 21 （DE3）.SDS-PAGE results showed that the fusion protein were expressed induced by 0.5 mmol/L IPTG,at 28 ℃ for 4 hours in E.coli BL21.And these soluble proteins could be purified by chromatography on nickel agarose column.Western blotting demonstrated that the recombinant fusion proteins can be recognized by anti-6-His monoclonal antibody.These results provide the basis for the studies of the physiological activity and the specific binding site and the function regulation of the MiRab11 protein.
出处
《热带作物学报》
CSCD
北大核心
2014年第9期1727-1732,共6页
Chinese Journal of Tropical Crops
基金
广西自然科学基金(No.2013GXNSFDA019011)
广西自然科学基金项目(No.2011GXNSFA018115)
关键词
芒果
MiRab11
突变体蛋白
原核表达
纯化
鉴定
Mango
MiRab11 gene
Mutant proteins
Prokaryotic expression
Protein purification
Identification
