Preparation Of Monoclonal Antibodies Against Escherichia Coli Heat-labile Enterotoxin B Subunit And Identification Of Linear B-cell Epitopes

Enterotoxin Escherichia coli (ETEC) is an important kind of virulence factors for causing diarrhea in babies and young pigs. After infection, they are dead of acute watery diarrhea and rapid dehydration. Has done great harm to human health and the aquaculture industry. The major virulence factors of ETEC including enterotoxin, adhesin, edema toxin, hemolysin and endotoxin. The enterotoxin including heat-labile toxin (LT) and heat-stable toxin (ST). The existing detection methods are ligated intestinal loops of adult rabbits and suckling mice gastric perfusion experiment, but not conducive to the rapid detection of enterotoxin. Therefore, study on monoclonal antibody and antigen epitopes of enterotoxin has important theoretical and practical significance for the establishment of rapid detection method, pathogenic mechanism and epitope vaccine development of enterotoxin.In this study, we generation of polyclonal and monoclonal antibodies against LTB and identification of linear B-cell epitopes on LTB.Preparation of polyclonal antibodies against LTB. Plasmids were extracted from strains DH5a/pET-30a-LTB, and transformed into Rosetta cells. The strain Rosetta/pET-30a-LTB induced to express by IPTG and obtained the recombinant LTB protein. The purified protein was used to immunize the rabbit and prepare polyclonal antibodies. The results of ELISA indicated that the titer of the serum reached1:1x218.Preparation of monoclonal antibodies (MAbs) against LTB. The eight-old BALB/C mice were immunized with the recombinant protein rLTB and the immuned spleen cells from these mice were fused with SP2/0myeloma cells. The supernatants of hybridoma cell were selected by indirect ELISA. Finally three hybridoma cells producing MAbs to LTB were acquired and named B1, D5, H10. The results of analyzing chromosome number displayed that the chromosome numbers of three hybridoma cell were from90to102. The isotypes of B1, D5, H10are IgG2b, IgG2b and IgG1, respectively. The supernatant titers of MAb B1, MAb D5, MAb H10were1:1×29,1:1×210and1:1×210, and the ascite titers were1:1.28x105,1:1.28x105and1:2.56×105. Blocking ELISA confirmed that only D5MAb was able to reacted with native LT. Western blot analysis showed that the resulting MAb D5had reacted specifically with LT and had no cross-reactivity to Heat-Stable enterotoxin (ST) and Shiga toxin (Stx). The results of neutralization tests indicated that the MAb D5was capable of neutralizing LT, with neutralization titer was1:50. Identification of linear B-cell epitopes on LTB. A set of10partially overlapping peptides covering the entire amino acid sequence of LTB protein (103amino acids) were synthesized. Western blot analysis results showed that the linear B-cell epitopes of LTB protein is located in the1-26aa、41-76aa and81-103aa. Antigenic epitope recognized by D5MAb lines in amino acid sequence of61-76and its sequence is61QKKAIERMKDTLRITY76.In conclusion, the obtained MAb D5was able to reacted specifically with the recombinant protein rLTB and native LT. It can provide a material basis for the diagnosis of LT. Antigenic epitope recognized by MAb D5also can provide the theoretical evidences for diagnosis methods based on epitope and epitope vaccine development.