膦丝菌素乙酰转移酶的表达及单克隆抗体制备

Preparation of monoclonal antibody against phosphinothricin acetyltransferase

目的表达有生物活性的膦丝菌素乙酰转移酶(PAT),制备PAT蛋白单克隆抗体(mAb)。方法通过PCR克隆bar基因编码区全长,将bar基因与原核表达载体pET28a(+)连接,构建重组质粒pET28-bar并转化大肠杆菌菌株BL21(DE3),经IPTG诱导目的蛋白表达,利用镍离子亲和层析纯化PAT蛋白,分析PAT蛋白活性。免疫BALB/c小鼠,取免疫后的小鼠脾脏与Sp2/0细胞融合,经间接ELISA筛选分泌抗PAT蛋白抗体杂交瘤细胞。结果在大肠杆菌中以可溶形式表达出重组PAT蛋白,纯化的重组PAT蛋白具有乙酰转移酶活性,制备了9株抗PAT蛋白mAb。结论表达的PAT蛋白可以用于除草剂解除剂研究,制备的mAb可能用于转基因产品的检测研究。

Objective To express phosphinothricin acetyltransferase （PAT） with biological activity and prepare monoclonal antibodies against PAT. Methods The full length bar gene was cloned by PCR and inserted into prokaryotic expression vec- tor pET28a（ ＋ ）. The recombinant plasmid pET28-bar was transformed into E. coil BI_2I （ 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. Results Soluble PAT was expressed in E. coil The purified PAT had the activity of acetyltransferase. We totally prepared 9 hybridoma cell lines which secreted specific anti-PAT monoclonal antibodies. Conclusion 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.