摘要
通过同源基因基因克隆、原核表达载体构建、GST融合蛋白诱导表达与纯化、新西兰大白兔免疫、抗血清分离和纯化制备了SPI多克隆抗体,并进行了玉米体内蛋白Western blot验证。结果表明,成功构建的PGEX-6T-1-SPI-C表达载体在大肠杆菌中表达并纯化了GST-SPI-C融合蛋白,通过免疫新西兰大白兔制备并纯化了SPI多克隆抗体。制备的SPI多克隆抗体不但能识别GST-SPI-C融合蛋白,而且能识别玉米体内全长SPI蛋白(112 kD),并具有较好的特异性。玉米子粒和胚乳的Western blot检测表明,SPI蛋白主要积累在胚乳中,与mRNA定量结果一致。
GST-SPI-C antigen was prepared by prokaryotic expression vector construction, expression, and purification of GST-SPI-C protein. With GST-SPI-C as antigen to immunize New Zealand white rabbits, Antiserum was isolated and purified from New Zealand white rabbit to prepare antibody successfully. The prepared SPI antibody was tested by western blot of maize protein in vitro and in vivo. The results showed that the successfully constructed PGEX-6 T-1-SPI-C expression vector. GST-SPI-C fusion protein was expressed and purified from E. coli.SPI polyclonal antibody was prepared and purified using GST-SPI-C fusion protein as antigen to immunize New Zealand white rabbits. The prepared SPI polyclonal antibody not only recognizes the GST-SPI-C fusion protein but also recognizes the full-length SPI protein(112 kD) in maize with excellent specificity. Western blot analysis of maize kernels and endosperm showed that SPI protein mainly accumulated in the endosperm, which was consistent with the quantitative results of mRNA.
作者
申蕾阳
青芸
吴楠
何姗
黄玉碧
余国武
SHEN Lei-yang;QIN Yun;WU Nan;HE Shan;HUANG Yu-bi;YU Guo-wu(College of Agronomy/Center for Crop Science Experimental Teaching,Sichuan Agricultural University,Chengdu 611130,China)
出处
《玉米科学》
CAS
CSCD
北大核心
2020年第1期104-110,共7页
Journal of Maize Sciences
基金
国家自然科学基金(31501322)
留学回国人员科技活动择优资助项目(00124300)
四川省博士后特别资助项目(03130104)
关键词
玉米
淀粉磷酸化酶
载体构建
蛋白纯化
抗体制备
Maize
Starch phosphorylase
Vector construction
Protein purification
Antibody preparation