Establishment And Reaserch Of Novel Immunoassay Methods For Bt Cry2Aa Toxin

Cry toxin is produced by Bacillus thuringiensis during sporulation.Cry2Aa toxin was first isolated in 1981.It is toxic to both Lepidoptera and Diptera.Due to the lack of Cry toxin receptors in mammals,Cry toxins are generally considered non-toxic to humans and livestock.Therefore,transgenic Bt plants have been widely cultivated in recent years.However,animals fed by genetically modified crops may cause genetically modified proteins to accumulate in mammals,and the flood of genetically modified plants will also destroy the diversity of the agricultural system and cause gene drift and other phenomena.It is necessary to establish a reliable,convenient and sensitive detection method for the expression level of Cry in transgenic Bt crops and the residue in food.In this paper,monoclonal antibodies and single-chain antibodies of Cry2Aa toxin were prepared,the corresponding immunological detection methods were established,and the actual samples of the lanes was tested.The research contented mainly include the following aspects:（1）Preparation of Cry2Aa toxin monoclonal antibodyThe prototoxin was used as the immune antigen,the Balb/c female mice were immunized by conventional immunization methods to obtain immune spleen cells of the mice,and then the spleen cells were fused with SP2/0 myeloma cells to screen monoclonal antibodies.The hybridoma cell line（1D2D11）with the highest sensitivity and stable secretion of Cry2Aa toxin antibody was selected to produce monoclonal antibody（m Ab）.After determination,the obtained monoclonal antibody was a highly specific antibody,the subtype is Ig G1 type,and the titer is 6.4×10⁵.It could be used as antibody material for subsequent establishment of Cry2Aa toxin immunoassay method.Cell strain 1D2D11（CCCCC NO:C2019188）was maintained by the China Typical Culture Preservation Center.（2）Construction of Cry2Aa toxin single-chain antibodyThe aforementioned hybridoma cell line 1D2D11 was used as the gene source,the heavy chain variable region（V_H）and light chain variable region（V_L）were amplified by PCR with universal primers,and then the entire gene sequence of single-chain antibody（sc Fv）was synthesized by SOE-PCR and sequenced.After the sc Fv gene was transferred into the vector PET-26b,it was expressed in E.coli BL21.The protein expression was identified by SDS-PAGE and Western blot,and the activity of sc Fv was identified by indirect ELISA.Subsequently,the successfully expressed sc Fv was biotinylated,and whether the labeling was successful was identified by SDS-PAGE and Western blot,and the biological activity of the acylated sc Fv was identified by the ligand binding test.The results showed that the VH and VL gene sequences were successfully amplified and the sc Fv gene sequence was constructed completely.After the sc Fv labeled successfully,the original biological activity was still maintained.It provides new antibody materials and new ideas for immunoassay technology.（3）Enzyme-linked immunoassay of Cry2Aa toxinThe Cry2Aa toxin ic-ELISA and DAS-ELISA method were established using the antibodies prepared above,and the working concentrations of the coating antigen and antibody were optimized.The standard curve of enzyme-linked immunosorbent method for the Cry2Aa toxin was established at the optimal concentration.In the ic-ELISA method,the inhibitory concentration(IC₅₀)was 10.65μg/m L,and the linear range(IC₂₀-IC₈₀)was 1.11-60.70μg/m L.This method had a certain cross-reaction with Cry1B（CR=8.8%）,and the cross-reaction with other Cry toxins was less than 0.1%.In the DAS-ELISA method,the values of LOD and LOQ were 10.76 and 20.70 ng/m L,respectively,and the cross-reaction rate with Cry1B was 3.1%.The values of SC₅₀ was 0.358μg/m L The three matrix effects of rice,corn,and soybean were optimized,and Cry2Aa toxin was added to the recovery rate test.The prepared sc Fv antibody was used to establish a DAS-ELISA method for Cry2Aa toxin.The corresponding standard curve was established at the optimal concentration of the capture antibody to detect the antibody.The values of LOD and LOQ were 118.75 and 633.48 ng/m L,respectively,which cross-reacted with Cry1B.The rate is 3.2%.The values of SC₅₀ was 0.749μg/m L.After confirming that the matrix effect was weakened or eliminated,an additive recovery test was carried out.The results showed that the sensitivity of DAS-ELISA is about 100 times higher than that of ic-ELISA in the detection method established by single resistance.The results of the ELISA were accurate and reliable,and could be applied to detect Cry2Aa toxins in genetically modified plants.（4）Chemiluminescence enzyme-linked immunoassay of Cry2Aa toxinIn the chemiluminescence system,the chemiluminescence enzyme-linked immunoassay（CLEIA）method was established by using the prepared monoclonal antibodies and single-chain antibodies.The concentration of capture antibody and detection antibody in this mode was optimized.When the monoclonal antibody was the detection antibody,the values of SC₅₀was 0.161μg/m L,the values of LOD and LOQ were 6.10 and 7.40 ng/m L,respectively,and the cross-reaction rate with Cry1B was 12.4%;when the single-chain antibody was used as the detection antibody,the values of SC₅₀ was 0.197μg/m L,the values of LOD and LOQ were 37.47 and 70.23 ng/m L,respectively.The cross-reaction rate with Cry1B was 19.3%.The recovery rate experiment was carried out in three substrates（rice,soybean and corn）.The results showed that the sensitivity of CLEIA was significantly improved compared with that of DAS-ELISA.The established CLEIA method had high reliability and accuracy and can be used for the rapid detection of Cry2Aa toxins in genetically modified plants.