The Establishment And Assay Of Elisa Method On Bt Protein In Transgenic Rice

The rapid development of study on insect-resistant plant gene engineering require more efficient assay method. Now the major methods include biology method (Laboratory test and Field test), DNA level test (PCR^ Southern blot) and protein level test (ELISA, Western blot). Each of the method has its own character. Compared with other methods, ELISA is the best for its fast, convenience and accuracy. The purpose of our research is to establish a set of complete ELISA method to assay the Bt content in transgenic rice.At first, protein CrylA(e) purified from bacteria was used as antigen to immune New Zealand rabbit. We successfully gained the antibody of CrylA(e) that had high liter. And we labeled the purified IgG with Alkaline Phosphatase by glutaraldehyde one-step method.We had experimented direct ELISA, indirect ELISA and double antibody-sandwich ELISA, concluded that the latter was a better method. And we had also confirmed the condition of the reaction.We had tested a lot of transgenic rice and proved that the content of Bt in the transgenic rice was related with its resistance on insect, without finding false outcome. At the same time, we had also tested the Bt content in different phases and organs, finding that the Bt content is different in different phases and different organs. We have also found many transgene silencing phenomena.