Gao Xudong, Wang Yongzhi, Shi Shengfeng, Li Zhongpeng, Li Xiaoyu, Xu Wenjing, Zhang Zhengkun, Lu Yang, Zhang Jiashi, Li Qiyun, Wang Jingang
College of Horticulture, Northeast Agricultural University, Harbin 150030, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 May;29(5):500-3.
To express phosphinothricin acetyltransferase (PAT) with biological activity and prepare monoclonal antibodies against PAT.
The full length bar gene was cloned by PCR and inserted into prokaryotic expression vector pET28a⁺. The recombinant plasmid pET28-bar was transformed into E.coli BL21(DE3), and under the induction of IPTG, PAT was expressed. The expressed protein was purified by Ni⁺; affinity chromatography to analyze its activity. The purified PAT was used to immunize BALB/c mice, and then the spleen cells from the immunized mice were fused with Sp2/0 cells. The hybridoma clones secreting antibodies against PAT were isolated by indirect ELISA and then subcloned.
Soluble PAT was expressed in E.coli. The purified PAT had the activity of acetyltransferase. We totally prepared 9 hybridoma cell lines which secreted specific anti-PAT monoclonal antibodies.
The expressed recombinant PAT can be used for biological reagent to prevent and relieve herbicide damage. Monoclonal antibodies against PAT may be used to detect the transgenic products.
表达具有生物活性的膦丝菌素乙酰转移酶（PAT）并制备抗PAT单克隆抗体。
通过PCR克隆bar基因全长，并将其插入原核表达载体pET28a⁺。将重组质粒pET28-bar转化至大肠杆菌BL21(DE3)中，在IPTG诱导下表达PAT。表达的蛋白通过Ni⁺亲和层析进行纯化以分析其活性。用纯化的PAT免疫BALB/c小鼠，然后将免疫小鼠的脾细胞与Sp2/0细胞进行融合。通过间接ELISA分离分泌抗PAT抗体的杂交瘤克隆，然后进行亚克隆。
在大肠杆菌中表达了可溶性PAT。纯化后的PAT具有乙酰转移酶活性。共制备了9株分泌特异性抗PAT单克隆抗体的杂交瘤细胞系。
表达的重组PAT可作为生物试剂用于预防和减轻除草剂损害。抗PAT单克隆抗体可用于检测转基因产品。
