C4植物PEPC酶的单克隆抗体制备和双夹心ELISA检测方法的建立  

Preparation of the Monoclonal Antibody Against C_4-Pepcase and Establishment of the Double Sandwich ELISA Detection Method

作　　者：张斌[1,2] 张启军[2] 廖慧敏[1,2] 秦海龙[1,2] 宗寿余[2] 夏士健[2] 吕川根[2] 

机构地区：[1]南京农业大学农学院,南京210095 [2]江苏省农业科学院粮食作物研究所,南京210014

出　　处：《分子植物育种》2015年第1期205-213,共9页Molecular Plant Breeding

基　　金：国家转基因重大专项(2011ZX08001004-009);江苏省农业科技自主创新资金(CX(12)1019)共同资助

摘　　要：磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPCase)是高等植物尤其是C4植物进行光合作用的一种关键酶,利用PEPC基因提高C3植物的光合效率具有重要的应用前景。本研究利用来源于玉米PEPCase免疫的BALB/c小鼠脾细胞与骨髓瘤细胞SP2/0融合,经筛选克隆,得到3株能稳定分泌抗PEPCase的单克隆抗体杂交瘤细胞,并制备腹水型单抗。利用其中一株杂交瘤细胞4E5的小鼠腹水单抗和PEPCase的兔源多抗,建立检测PEPCase的多抗一待检样品一单抗模式的双夹心酶联免疫吸附检测方法(enzyme linked immuno sorbent assay,ELISA)。检测条件经过优化后分别为:兔源多抗以1:3 200稀释,单抗4E5以1:800稀释,整个操作过程仅需要5个小时(不含包被和封闭时间),本方法能够长期保存。利用建立的双夹心ELISA检测方法检测验证转玉米PEPC基因水稻,在经PCR分子检测后证实携带PEPC基因的305株样品中,有237株为阳性单株,阳性率为77.7%,符合率较高。实验表明,本研究能够为育种上快速筛选转PEPC基因作物提供了一种更加简单、快速、灵敏的检测方法。Phosphoenolpyruvate carboxylase （PEPCase） is a key enzyme during photosynthesis in C4 plant. Introd- ucing PEPC gene into C3 plant is of great importance to improve its photosynthesis efficiency. In this study, three positive hybridomas cell lines secreting the monoclonal antibodies （McAb） against PEPCase were obtained by the fusing mouse myeloma cells （SP2/0） with spleen cells from BALB/c mice immunized with PEPCase from maize. The 4E5 McAb and the rabbit polyclonal antibody was prepared and purified to establish the double sandwich ELISA for detecting PEPCase. We optimized the condition of this double sandwich ELISA： the rabbit polyclonal antibody and the 4E5 McAb were diluted in ratio of 1：3 200 and 1：800, respectively. Only 5 hours were enough for the whole process （coating and blocking time were not included）, and the monoclonal antibody could be preserved for a long time. Among 305 samples selected by the molecular approaches, 237 positive individuals were identified using the optimized double sandwich ELISA. The coincidence rate of two methods was about 77.7%. The results suggested that the double sandwich ELISA method should be a rapid, sensitive, economic and specifictool to detect the PEPCase for breeding the PEPC gene modified rice.

关 键 词：C4植物 PEPCase 单克隆抗体 双夹心酶联免疫吸附试验 

分 类 号：Q943[生物学—植物学]