CAJ | 학술논문

建立并优化了转基因油菜(Brassica campestris)中的PAT蛋白酶联免疫检测技术.首先通过Western杂交和酶活性分析鉴定了PAT蛋白的纯度和活性.然后以其为抗原免疫新西兰大白兔制备了PAT蛋白的多克隆抗体,并采用硫铵沉淀法和protein A-Sepharose 4B对其进行了纯化.所得抗体对PAT蛋白的检测限为2×10-5 mg/mL,且与植物源的几种结构功能相关蛋白均无交叉反应.然后对植物蛋白进行初步提取,应用酶联免疫检测技术对转基因油菜(MS1/RF1 and MS8/RF3)中的PAT蛋白进行了检测,结果表明ELISA能高效地鉴别转基因和非转基因油菜.
건립병우화료전기인유채(Brassica campestris)중적PAT단백매련면역검측기술.수선통과Western잡교화매활성분석감정료PAT단백적순도화활성.연후이기위항원면역신서란대백토제비료PAT단백적다극륭항체,병채용류안침정법화protein A-Sepharose 4B대기진행료순화.소득항체대PAT단백적검측한위2×10-5 mg/mL,차여식물원적궤충결구공능상관단백균무교차반응.연후대식물단백진행초보제취,응용매련면역검측기술대전기인유채(MS1/RF1 and MS8/RF3)중적PAT단백진행료검측,결과표명ELISA능고효지감별전기인화비전기인유채.
The immunoassay method to detect genetically modified (GM) rape (Brassica campestris ) containing the phosphinothricin acetyltransferase (PAT) were developed and applied. The purified PAT was identified by Western blot and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both saturated ammonium sulfate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of polyclonal antibody has been demonstrated in ELISA test.The result of the ELISA for antiserum sensitivity was about 2× 10-5 mg/mL and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1%. The detections of transgenic plants were evaluated with two transgenic rapes (MS1/RF1 and MS8/RF3) and the transgenic rapes (MS1/RF1 and MS8/RF3) could be easily distinguished by ELISA.