Expression Of The Subunits Of Heat-labile Enterotoxin Of Enterotoxigenic Escherichia Coli And Preparation Of Monoclonal Antibodies Against LT

Heat-labile enterotoixin (LT), produced by Enterotoxigenic Eshchirichia coli (ETEC), is the virulence factor of diarrhea in humans and animals. More interesting, LT also possesses strong immunogenicity and can assist other antigens delivered through mucosal pathway to produce specific antibodies, so it is a good mucosal immunization adjuvant and has significant medical and economic values in mucosal immunization research and development of vaccine for mucosal immunization.In this research, to extract toxin, E. coli reference strain 44814 was cultured on CAYE-2 medium, and the collected bacteria were treated with polymixin B and followed by ultrasonic process. The enterotoxin extraction were obtained by precipitating the supernatant by using ammonium sulfate and removing the salts with"D-Salt Dextran Desalting Column". The following tests on rabbit and CHO cells indicated that the enterotoxin extraction could lead the intestinal epithelium overmuch excrete and induce CHO cells distortion or death. The results confirmed that the extraction were contained heat-labile enterotoxin.To study the biology characteristic of LT, two pairs of primers specific to LTA and LTB were designed and synthesized according to the published sequence of LT genes. Following the genes of lt-A and lt-B were amplified from strain 44814. The two fragments were subcloned into expression vector pGEX-6p-1. The resulting plasmid contained the lt-A gene was designed as pGEX-LTA and the plasmid contained the lt-B gene was designed as pGEX-LTB. By SDS-PAGE analysis and Western blotting, it confirmed that BL21 E.coli which contained pGEX-LTA was able to express large quantities of a 56-ku fusion protein (GST-LTA), and BL21 E.coli which contained pGEX-LTB was able to express large quantities of a 39-ku fusion protein (GST-LTB).In order to identify Heat-labile enterotoixin, it is necessary to have the monoclonal antibody . The fusion protein GST-LTA was immunized the 6 weeks old BALB/c mice 4 times. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method, and the interested antibody were detected by indirect-ELISA and Dot-ELISA methods. The hybridoma cell strain F8 and G9 were obtained, which can stably secret monoclonal antibody specific for LT-A. Further more, indirect ELISA by using the monoclonal antibodies were performed to detect LT, and made a basis for further setting up a LT-testing method which has high specificity and sensibility, more over can be operated easily and quickly.