Development Of Enzyme-linked Immunosorbent Assay (ELISA) For Determination Of Transgenic Bt-cotton

The goal of this research was to study on the preparation of antigen of Bt (Bacillus Thuringiensis) insecticidal crystal protein and a sandwich enzyme-linked immunosorbent assay (ELISA) was developed and performed to determine the Bt Cry1 Ac protein in cotton.Three species of Bt (HD-1, HD-2, HD-73) were cultured in two conditions (28 ℃, 150r/minand28 ℃, 200r/min)by two kinds of medium (1/2LB,1/2NB), the thallus were treated with lysis and then were dealed with isoelectric point deposition and high-speed centrifugation respectively. Result indicated that the yield of Bt protein was very high when three species were fermented in 1/2NB medium, 28 ℃, 200r/min for 3 days. Although the quantity of Bt protein for high-speed centrifugation was lower than that for isoelectric point deposition, it had,higher purity. Therefore, the Cry1Ac protein which produced only enriched by HD-73 was selected as antigen for developing of polyclonal antibodies.In this experiment two methods for antibody preparation were adopted, the first one used solubilization of protein sample in buffer as immunogen and the second one used emulsification of protein band obtained from polyacrylamide gels as immunogen. Results showed that the rabbits immunized by the second method showed intenser immune response in the course of immunity, however, the lower-titer antibodies was obtained compared with the first ones and antibodies from animals immunized by firsr one were chosen for ELISA and acted as the first antibody. The secondary antibody was labeled with horseradish peroxidase (HRP). The limit of detection was 0.005 ug/mL with a linear range approxiamately from 0.015-0.25 μg/ml and there was no cross-reaction with other Bt Cry proteins (Cry1C, Cry2A, Cry3A, Cry3Bb1, Cry9C).The assay was applied to determine six different cottons (GK-12, GK-22, NON, insect-resistant cotton, bivalent transgenic cotton, shiyuan 321). Three different buffers (Tris-borate buffer, sodium carbonate buffer, PBST buffer) were used for protein extraction and results showed Tris-borate buffer was the best. Meanwhile, for the validation of the ELISA test, cotton leaf samples were analyzed by polymerase chain reaction (PCR) method. The primer pairs CaMV-35S (Cauliflower Mosaic Virus, CaMV-35S), Cry1Ab + Cry1Ac, Cry1Ac as detection gene and 18S rRNA as inner gene were selected, and results by agarose gel electrophoresis showed that GK-12, GK-22, insect-resistant cotton, bivalent transgenic cotton and shiyuan 321 were positive samples and NON was negative...