Study On The Characteristics Of K88and LTb Via Intranasal Vaccination To Mice

Enterotoxigenic Escherichia coli (ETEC) infections are a significantcause of diarrheal disease in young stock. According to statistics,about35%of piglet diarrhea is coused by ETEC in domestic,and more than45%of newborn piglet diarrhea is coused by ETEC in America. ETECcaused diarrhea have high morbidity and mortality. It caused very seriouseconomic losses to the livestock industry.Currently, many countriesdedicated to the development of new effective vaccines to preventdiarrhea caused by ETEC.ETEC pathogenic factor includes enterotoxins and fimbriae. Whenpathogens infect host,ETEC stick to small intestinal mucosal epithelialcell,then settle and reproduce. The ETEC release enterotoxin which cancombine with specific receptors on the cell membrane of smallintestine,then it couse intracellular water and electrolyte imbalance andcell absorption barriers。 Eventually，it lead to the occurrence ofdiarrhea。There are mainly7types of ETEC adhesin in pigs: K88、K99、987P、F17，F18、F41、F42, of which, diarrhea among piglets was mainlycaused by K88. Enterotoxin includes heat-stable and heat-labile enterotoxin.LTb, a subunit of heat-labile enterotoxin of E. coli, is widely used byvirtue of its non-toxicity and effectiveness as a mucosal adjuvant.Effective immune response could be induced via nasal mucosa where richblood vessels and lymphoid tissue lie in. In this study, in hope ofstimulating specific mucosal and systematic immune response, BALB/cmice were vaccinated via intranasal vaccination. We hope this researchwill bring a new mthod in developing safe,effective and convenientgenetic-engineered vaccine against ETEC.Objective: We aimed to construct the K88fimbrial protein of ETECand the Escherichia coli heat-labile enterotoxin B subunit (LTb) into thevector of prokaryotic expression and got the purified recombinantproteins.Then observed the immunopotency of the K88and LTb afterintranasal vaccination to mice.Methods: K88and LTB were cloned by polymerase chain reaction(PCR) and constructed into an expression vector pQE30, respectively.The recombinant K88and LTb were expressed in Escherichia coli M15.Recombinant proteins were purified and renatured, then BALB/c micewere administered via nasal route with recombinant K88together with orwithout LTb. After four dosages of vaccination, the antibody levels wereanalysed by enzyme linked immunosorbent assay (ELISA).Results: K88and LTb were expressed by E.coli M15and the purity of recombinant proteins were up to95%after affinity chromatography.Compared to K88group and negative control group, the mice vaccinationwith K88plus LTB group reached a significant K88-specific serum IgGantibody and K88-specific mucosal sIgA antibody in the nasal cavity andsmall intestine (p<0.01).Conclusion: Playing a role of adjuvant, LTB intranasal immunizationwith K88can confer BALB/c mice to increasedly produce both serumIgG antibody and mucosal sIgA antibody. The results may be useful indeveloping new ETEC genetically engineering vaccine.