耐草甘膦基因G6原核表达蛋白条件的优化及其免疫反应性分析

Optimization on prokaryotic expression conditions of glyphosate: resistant G6 gene and its immunoreactivity

目的在大肠杆菌中表达耐草甘膦基因G6,并对其进行免疫反应性分析。方法将已构建好的携带G6基因的重组质粒pET28a-G6转化到大肠杆菌BL21中诱导表达,分别从诱导温度、异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度及诱导时间进行了表达条件的探索。获得的重组蛋白在非变性条件下镍亲和层析纯化后,经质谱鉴定,对其免疫反应活性进行测定,分析该原核表达蛋白与转G6基因水稻中蛋白的等同性。结果利用原核表达系统成功实现了G6重组蛋白的高效表达,其最适条件为:0.5 mmol/L IPTG、30℃和160 r/min摇床转速诱导7 h。质谱鉴定为5-烯醇式丙酮酸-莽草酸-3-磷酸合成酶(EPSPS)。Western blot鉴定后,表达的重组蛋白与转G6基因水稻中的G6蛋白有相同的免疫反应性。结论利用原核表达系统表达的G6重组蛋白可以替代植物外源蛋白进行转G6基因产品的食用安全性评价,也可用于转G6基因产品ELISA检测的抗体制备。

Objective To optimize the Bacillus coli expression condition of gly- phosate-resistant G6 gene, and analysis its immunoreactivity. Methods The recombinant vector PET28a-G6 was transformed into BL21 and its expression condition, such as temperature, IPTG and time, were optimized. Then Ni-NTA affinity chromatography was used to purify the recombinant protein. After mass spectrum identification, the equivalence of G6 proteins expressed in Bacillus coli and in transgenic G6 rice was analyzed by immune recognition. Results The successful expression of G6 recombinant was achieved by using prokaryotic expression system. The optimum condition of expressing G6 was 0.5 mmol/L IPTG, 30 ~C and 160 r/min, 7 h, and the protein was identified as EPSPS by mass spectrum. G6 expressed protein in Bacillus coli was substantially equivalent to that of rice genetically modified with G6 in tests of immune recognition on western blot. Conclusion This protein can be used for food safety assessment, detection of genetically modified products with G6 gene and for the preparation of antibody in ELISA detection.