不同样品前处理对转基因棉Bt蛋白含量测定的影响

Effects of Different Samples Pre-treatment on Determination of Protein Content in Transgenic Bt Cotton

[目的]寻找一种稳定、准确、灵敏度高的检测转Bt植物中Bt杀虫蛋白的方法。[方法]比较几种ELISA方法检测Bt杀虫蛋白的效果,并就样品前处理方式以及温育时间、显色时间等影响因子进行分析。[结果]自制抗体法较试剂盒更为灵敏,其同一时期同一组织的Bt蛋白含量显著高于试剂盒,但不同方法对Bt蛋白含量的检测结果在时空表达变化的趋势上基本一致。样品在液氮研磨后抽提5或8 h,对检测结果影响较小,差异未达显著水平;匀浆机研磨过滤后离心与否,对检测结果影响较小。从样品前处理方式看,匀浆机较液氮研磨对样品组织蛋白的萃取效率更高,其检测结果显著高于液氮研磨处理的样品,差异达显著水平。温育时间和显色时间的延长会增加样品的吸光值,对蛋白含量检测值有一定影响,但差异不显著。[结论]不同样品前处理方式对转基因棉Bt蛋白含量测定存在显著影响。

[ Objective ] The aim was to seek a stable, accurate and high sensitive detection method for Bt insecticidal protein in transgenic Bt plants. [ Method] The detection effect of several commonly used ELISA methods for Bt insecticidal protein were compared ,pre-treatment methods and influencing factors such as incubation and coloration time were analyzed. [ Result] The homemade antibody method was more sensitive than the kit,which measured Bt protein content in the same period and organization was significantly higher than the kit,but the detection results were basically the same in spatial-temporal expression change. After grinding the samples in liquid nitrogen and extracting for 5 or 8 h,there were less impact on the test results,the difference was not significant; homogenized and filtered centrifugal grinding machine or not,had little effect on the test results. From sample pre-treatment methods,the higher extraction efficiency compared to liquid nitrogen grinding machine homogenized tissue protein samples,the test results were significantly higher than the sample in liquid nitrogen grinding treatment,the difference was significant. Prolong of incubation and coloration would inci＇ease the sample abserbance values, and have a certain effect on the protein content detected values, but the difference was not significant. [ Conclusion] Different samples pre-treatment has significant effects on determination of Bt insecticidal pretein in transgenic Bt plants.