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Construction Of RBL-2H3 Cell Line Stably Expressing Calcium Probe And Preparation Of Monoclonal Antibody Against Rat IgE-Fc Fragment

Posted on:2021-03-22Degree:MasterType:Thesis
Country:ChinaCandidate:Z E MaFull Text:PDF
GTID:2370330602478318Subject:Food engineering
Abstract/Summary:PDF Full Text Request
There are a large number of Fc?R I receptors on the surface of RBL-2H3 cells,which provide favorable preconditions for the construction of cell sensors based on IgE specific recognition and various signal factors released by cell degranulation.However,the content of traditional IgE antibody in animal body is rare and preparation is difficult.In addition,Ca2+concentration,which is widely used as the characteristic signal of degranulation,is also faced with the defects of high cost and complicated operation steps because it is mostly based on the traditional fluorescent probe.In this study,RBL-2H3 cell line with stable expression of calcium ion probe can avoid directly using the traditional fluorescent probe to dynamically characterize the change of Ca2+concentration;in order to solve the problem of traditional IgE antibody preparation difficulty,some researchers proposed the path of preparing IgE chimeric antibody.However,one of the key steps in the preparation of IgE chimeric antibody is the need for antibodies that specifically recognize the fragment of IgE-Fc.In this study,monoclonal antibodies against IgE-Fc fragment were prepared based on monoclonal antibody technology,which provided a useful characterization tool for the construction of IgE chimeric antibody.The above results will provide favorable support for the construction of RBL sensor.The results of this study are as follows:1)RBL-2H3 cell line was constructed by liposome transfection of PYC3.60-C1 plasmid which can express cameleon of Ca2+sensitive protein.The optimal sensitization concentration of C48/80 on RBL-2H3 cells was 150 ?g/mL,and the release rate of ?-HEX was 55.7%.The results showed that when the concentration of C48/80 increased to 100 ?g/mL,150?g/mL and 200 ?g/mL,the relative fluorescence intensity of RBL-2H3 cells increased synchronously within 50s,and decreased gradually after 1 min,which was similar to the signal indicated by the traditional Fluoro-4/Am fluorescence probe.The stable cameleon RBL-2H3 cell line can be used to detect the degranulation of sensitized cells in real time by the calcium ion probe cameleon contained in the cell itself without adding a probe.2)The recombinant rat IgE-Fc fragment was used as immunogen to immunize Balb/c mice,and the No.1 mouse with the best immune effect was selected for hybridoma cell fusion.The results showed that the fusion rate of cells was 83.68%.Seven positive clone hybridoma cell lines with stable secretion of anti rat IgE-Fc antibody were obtained by indirect ELISA combined with limited dilution subcloning.They were numbered 1D10,4A4,4E7,5C11,6E4,6E8 and 7B6 respectively.After the positive clone hybridoma cell lines were expanded and cultured,they were injected into the abdominal cavity of mice respectively to collect ascites.After the crude purification by ammonium octanoate sulfate method,seven kinds of monoclonal antibodies against rat IgE-Fc were obtained.The results of indirect ELISA showed that the ascites titers of 1D10,4E7,5C11 and 7B6 were all 1:128000,and the titers of 5C11 were the highest compared with the other six,reaching 1:1400.The results of specificity analysis showed that the above seven monoclonal antibodies were specific for IgE-Fc of rats,and there was no significant cross reaction with ?-lactamase,OVA and BSA Shall have good binding characteristics.
Keywords/Search Tags:RBL-2H3 cell, Cameleon, IgE in rats, Monoclonal antibody
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