抗草胺膦bar基因原核表达和纯化及其免疫反应性分析

Prokaryotic expression,purification and immunoreactivity analysis of glufosinate resistant gene(bar)

目的实现抗草胺膦bar基因在原核表达系统中的高效表达及纯化,并分析其免疫反应性。方法将携带bar基因的重组质粒pET28a-bar转化到大肠杆菌BL21(DE3)中诱导表达,确定最佳诱导表达条件。获得的重组蛋白经镍亲和层析纯化后,免疫BALB/c小鼠,测定抗体效价,以获得的抗BAR的抗血清作为一抗进行Western blot反应,检测蛋白的免疫反应性,分析该原核表达蛋白与转bar基因油菜中蛋白的等同性。结果利用原核表达系统成功实现了BAR重组蛋白的高效表达,其最适表达条件为:0.5 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)、20℃和120 r/min摇床转速诱导过夜。以纯化后的目的蛋白作为抗原免疫小鼠,得到抗BAR的抗血清。以间接ELISA法测定抗血清效价达1∶102 400,将其作为一抗进行Western blot反应,结果显示该抗体与原核表达的重组蛋白及转bar基因油菜中的BAR蛋白均能特异性结合。结论获得了高纯度的BAR重组蛋白,制备了特异性抗BAR多克隆抗体。原核表达的重组蛋白与转bar基因油菜中的BAR蛋白具有相同的免疫反应性,为进行转bar基因产品的食用安全性评价提供实验依据。

Objective To obtain high expression of bar gene in Eseherichia coli and purify its expressed protein, then analyze its immunoreactivity. Methods The recombinant vector pET28a-bar was transformed into BL21 and its expression condition was optimized through temperature and IPTG, Ni-NTA affinity chromatography was used to purify the recombinant protein. The purified protein was used to immunize BALB/c mice, the titer of antiserum was tested by ELISA. The equivalence of BAR protein expressed in Escherichia coli and in transgenic bar rape was analyzed by Western blot. Results The prokaryotic expression system was used to express BAR recombinant protein. The optimum condition of expressing BAR was 0.5 mmol/L IPTG, 20 ℃ and 120 r/min, overnight. The titer of the antiserum from immunized BALB/c was 1：102 400 by indirect ELISA. Western blot showed that antiserum could bond specially with BAR recombinant protein and transgenic bar rape. Conclusion Specific polyclonal antibody with high purified BAR recombinant protein has been prepared in the present study and the results will provide the basis for food safety assessment of genetically modified products with bar gene.