Construction And Expression Of Anti-Cry1Ac Protein IgE Chimeric Antibody And Its Application In Biosensors

Cry1Ac is a parasporal crystal protein of Bacillus thuringiensis which widely exists in the soil.It has insecticidal activity against different kinds of insects and is called Insecticidal Crystal Protein?ICP?.At present,Cry1Ac has been widely used in the field of prevention and control of transgenic crop biological pests.The establishment of a rapid and sensitive analytical method for the genetically modified crops is an important direction in the field of food safety research.The starting point of this study is to construct a RBL cell sensor that can be applied in the field of food safety analysis,aiming at the key scientific problem of the difficulty in the preparing the core element IgE of the RBL cell sensor,the Cry1Ac protein was used as the research object,On the basis of the early work of obtaining the specific Cry1Ac protein nanobody?VHH?,the nanobody was used as a core recognition element,and was fused with the Fc fragment of Rat IgE,to construct a VHH-Fc chimeric IgE antibody against Cry1Ac by the gene engineering technology.In the hope of future IgE chimeric antibody was used as a substitute of traditional IgE antibodies for the application of RBL cell sensor system.The main experimental results of this study were as follows:1.Through gene engineering technology,anti-Cry1Ac nanobodies N24 and N72were fused with the Fc fragment?C?2C?3C?4?of Rat IgE by using Flexible Linker F?GGGGSGGGGSGGGGS?and Rigid Linker R?PAPAP?,to construct a IgE chimeric antibodyexpressionvectors?pPIC9K-N72-F-Fc,pET25b-N72-F-Fc,pET25b-N72-R-Fc,and pET25b-N24-R-Fc?of nanobody-based recognition element.The sequencing results showed that the foreign genes of encoding anti Cry1Ac protein IgE chimeric antibodies were inserted into the pPIC9K and pET25b?+?vectors in the correct sequence,respectively,and no base mutations were found.2.The pPIC9K-N72-F-Fc expression vector was transformed into the eukaryotic expression system Pichia pastoris GS115 and the expression of IgE chimeric antibody was carried out.After induced with methanol,the medium supernatant were extracted,SDS-PAGE electrophoresis analysis showed that no obvious target protein band was detected in the expected expression supernatant.DAS-ELISA assay was no significant difference in OD values with BSA and OVA negative controls,and no expression of N72-F-Fc chimeric antibody was detected.3.The three IgE chimeric antibody expression vectors?pET25b-N72-F-Fc,pET25b-N72-R-Fc and pET25b-N24-R-Fc?were transformed into the prokaryotic expression system E.coli.Rosetta?DE3?.After 10 hours of induction at 30°C,180r/min and 0.1 mM IPTG,purified obtain three higher purity anti-Cry1Ac protein IgE chimeric antibodies?N72-F-Fc,N72-R-Fc and N24-R-Fc?by Ni²⁺affinity column.4.After denaturation,purification and renaturation of N72-F-Fc,N72-R-Fc and N24-R-Fc,the IgE chimeric antibody terminal signal and the binding performance with Cry1Ac antigen were analyzed by ELISA.The results showed that both the His-tag and the IgE-Fc fragment of the IgE chimeric antibody retained activity,and the binding properties of N24-R-Fc and Cry1Ac were strong and specific.A standard curve of DAS-ELISA was established using N24-R-Fc as the capture antibody and8A8 as the detection antibody to detection of Cry1Ac.The linear detection range was3.9-125 ng/mL,and a limit of detection was 2.58 ng/mL.The effects of different concentrations of N24-R-Fc chimeric antibody on the activation and degranulation of RBL-2H3 cells induced by 5?g/mL of Cry1Ac antigen were preliminary analyzed.The results showed that:With the increase of N24-R-Fc antibody concentration,the degree of degranulation of RBL-2H3 cells gradually increased.Under conditions of20,30,40,and 50?g/mL of N24-R-Fc chimeric antibody,The release rates of RBL-2H3 cells degranulation?-HEX were 11%,18.7%,28.3%,and 33.3%,respectively.preliminary showed that the N24-R-Fc chimeric antibody can be used as a substitute for traditional IgE antibodies and was applied to the RBL cell sensor system.5.A label free impedance immunosensor was established for Cry1Ac using N24-R-Fc chimeric antibody and anti-Cry1Ac monoclonal antibody?8A8?as recognition elements,respectively.The results showed that the linear ranges of Cry1Ac detected based on N24-R-Fc and 8A8 Cry1Ac impedance immunosensors were 1.95-62.5 ng/mL,0.98-125 ng/mL,respectively.and a limit of detection?LOD?of 1.85 ng/mL,0.80 ng/mL,respectively.Using N24-R-Fc based Cry1Ac label free impedance immunosensor for corn spiked recovery experiments,the recovery rate was 87.32-116.11%,CV?%?was less than 12%.The results showed that the method had good accuracy.