APPLICATION OF STEPWISE AMMONIUM SULFATE PRECIPITATION AS CLEANUP TOOL FOR AN ENZYME-LINKED IMMUNOSORBENT ASSAY OF GLYPHOSATE OXIDOREDUCTASE IN GENETICALLY MODIFIED RAPE OF GT73

Abstract

ABSTRACT The method of enzyme-linked immunosorbent assay after stepwise ammonium sulfate (AS) purification (AS-ELISA) was developed and used to detect genetically modified (GM) rape of GT73 containing glyphosate oxidoreductase (Gox). Gox protein encoded by the Gox gene from Achromobacter sp. was highly expressed as inclusion bodies in Escherichia coli BL21 (DE3) and purified to homogeneity by Ni2+affinity chromatography. A simple and efficient extraction and purification procedure of Gox protein from the seeds and leaves of GM rape was developed by means of stepwise AS precipitation. Purified polyclonal antibodies against Gox was produced and enzyme-linked immunosorbent assay (ELISA) procedures were established further on to measure the Gox protein. AS-ELISA allowed 5% GMOs to be detected in the seeds of GT73 and 0.5% GMOs to be detected in the leaves of GT73 rape, which makes this method an acceptable method to access Gox protein in GM rape of GT73. PRACTICAL APPLICATIONS Many GMOs containing Gox gene have been approved worldwide such as GT73 rape, 1,445 cotton and Mon832 maize. Protein based methods were more important than DNA based methods, because protein performs a specific and concrete function and is closely interconnected with crop traits. AS-ELISA method can be used in the screening of GM plant, Gox protein expression assay and quantitative detection for GMO labeling. AS-ELISA Gox detecting method was established in this paper and was being evaluated of Inter-laboratory Comparison in some of Chinese GMO detection and assessment centers. With the knowledge of ELISA, ELISA method will be the national standards and international and will be a beneficial supplement for the DNA based GMO detecting methods.