摘要
【目的】在大肠杆菌系统中原核表达拟南芥叶绿体Mg^(2+)转运蛋白AtMGT10,通过免疫健康成年家兔,获得针对AtMGT10蛋白的多克隆抗体,为研究AtMGT10蛋白在植物生长发育中的功能奠定基础。【方法】利用PCR技术克隆AtMGT10基因编码区187~786bp的cDNA片段,连接到pET-28a中构建原核表达载体pET-28aAtMGT10^(63-262),转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达带有His标签的His-AtMGT10^(63-262)截短重组蛋白。用镍柱亲和纯化后的重组蛋白作为抗原皮下注射免疫家兔,制备针对AtMGT10蛋白的多抗血清。提取拟南芥野生型叶片总蛋白,用AtMGT10多抗血清进行蛋白免疫印迹检测。【结果】经PCR扩增获得了600bp的AtMGT10基因cDNA片段,成功构建原核表达载体pET-28a-AtMGT10^(63-262),在大肠杆菌系统中表达出His-AtMGT10^(63-262)蛋白,经亲和纯化后免疫家兔获得了Anti-AtMGT10多抗血清。在拟南芥野生型总蛋白中用Anti-AtMGT10经免疫印迹检测到大约50ku的蛋白条带,利用抗原竞争试验进一步证明检测到的信号就是拟南芥内源AtMGT10蛋白。【结论】原核表达了His-AtMGT1063-262蛋白,成功制备了特异性较强的AtMGT10蛋白多克隆抗体。
[Objective] The objective of this study was to heterologously express Arabidopsis thaliana magnesium transporter 10 （AtMGT10） in Escherichia coli and to generate polyclonal antibody against At- MGT 10 in rabbits using recombinant AtMGT10 as the antigen. This work would lay foundation for the study of AtMGT10＇s role in plant growth and development. [Method] Partial AtMGTIO cDNA fragment （187-786 bp） was cloned into pET-28a vector. The resulted product, pET-28a-AtMGT1063-262, was trans- formed into E. coli strain BL21 （DE3） for expression of the recombinant protein His-AtMGT1063-262. His- AtMGT1063-262 was then purified via nickel affinity chromatography and used to immune rabbits to obtain antiserum against AtMGT10. Total proteins were also extracted from wild type Arabidopsis leaves and subjected to western blot analysis to test whether endogenous AtMGT10 can be detected by AtMGT10 an- tibody. [Result] AtMGTIO cDNA fragment （187--786 bp） was cloned and used to construct pET-28a-At-MGT1063262 expression vector. His-AtMGT1063~z62 was successfully expressed in E. coli BL21 （DE3） and purified to immune rabbits. Anti-AtMGT10 antiserum was obtained and used to detect endogenous At- MGT10 in wild type leaf protein samples. Competition test confirmed that a 50 ku band in Arabidopsis to- tal protein samples detected by Anti-AtMGT10 was the endogenous AtMGT10 protein. [Conclusion] His- AtMGT1063-262 was expressed and the polyclonal antibody against AtMGT10 protein was prepared success- fully.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2017年第5期168-174,共7页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31300988)
中央高校基本科研业务费项目(2452015215)
教育部博士点基金新教师项目(20120204120036)
