The Construction Of The Mutant Escherichia Coli Heat-labile Enterotoxin And Its Immune Adjuvant Activity On Mucosa And The Preparation Of Monoclonal Antibodies Against LTB

Enterotoxic Escherichia coli (ETEC), the most common and imporant pathogenic E. coli, can cause the diarrhea in humans and young stocks. It is common that Primary young stocks are dead of violent water sample diarrhea and fast dehydration after the infection. The disease incidence and case fatality are both highly. LT, produced by Enterotoxic Escherichia coli (ETEC), is the virulence factor of diarrhea in humans and animals. LT, a hexmeric protein, is composed of one A subunit with ADP-ribosylating activity, and the homopentameric B subunit containing the monosialogangliosied (GM1) receptor-binding site. Although LT has a potent toxicity, it possesses strong immunogenicity and has an effect of mucosa immunological adjuvant activity which can help foreign antigen to induce specific mucosa immunological response. Thus, LT has significant medical and economic values in mucosal immunization research and development of vaccine for mucosal immunization.To construct non-toxic mutant of LT retaining the mucosa immunological adjuvant activity as mucosal asjuvant for many other antigens with the aim to supply a basis for the study of mechanism of LT and its use in vaccines, in this research, A genetically modified LT-A subunit was generated by mutation at residues 63 (Ser-Lys) using the PCR. The PCR product was cloned into prokaryotic expression vector pGEX-6p-1 and resulting recombinants was called pGLT-Amu respectively. The recombinant plasmid was transformed into E. coli BL21 and GST-fusion protein with expected size was expressed by Western blotting using monoclonal antibody specific for LT-A.To determine the effects of sonicated E. coli expressing the mutated A subunit of LT on immune response, 80 healthy ten-day-old chickens were assigned to 4 groups. The first group and the second group were orally immunized by the LaSota live vaccine against NDV with different dose of LT mutant as adjuvants. The third group was orally immunized by the LaSota live vaccine with normal saline water. And nothing has been done on the fourth group as control. On the 7th, 14th, 21st, 28th day after the immunization, the antibody specific for NDV was determined. It was showed that the recombinant mutant could improve the hemagglutination inhibition antibody titer and mucosa antibody titer. Meanwhile, in this research, the fusion protein GST-LTB was immunized the six-week-old BALB/c mice 4 times. The splenocytes of immunized mice were detected by indirect-ELISA method. The hybridoma cell strain G5 and F7 were obtained, which can stably secret monoclonal antibody specific for LT-B. Further more, indirect ELISA by using the monoclonal antibodies were performed to detect LT, and make a basis for further setting up a LT-testing method with high specificity and sensibility, which makes test be done easily and quickly.