Construction Of Double Targeting Lactobacillus Casei Expression F4~+ETEC And Immunogenicity Analysis

Enterotoxigenic Escherichia coli(ETEC)is an important pathogen of newborn and weaning piglets diarrhea,and F4+ enterotoxigenic Escherichia coli produce the greatest impact on them.For this reason,prevention and control of F4+ ETEC to infect piglets become a key ring to prevent and cure diarrhea.For the economic problems of long-term effects of diarrhea to the farming households,in this study,we use lactobacilli genetically engineered vaccine,with the dendritic cells and M cells targeting peptides as an adjuvant molecules to produce effective oral mucosal immunity in gastrointestinal tract to form an effective first defense line to resist the damage produced by enterotoxigenic Escherichia coli in newborn and weaned pigs.At the same time,we explore the influence and regulation of dendritic cells and M cells targeting peptide to the mucous membrane immune system.In this study,we use the fusion gene F4+ ETEC STa-LTB-STb(K)constructed and preserved in our laboratory,which is the protective antigen gene of F4+ ETEC,on the basis of gene K,add M cells targeting peptide col at one end of gene K to get gene KC to target M cell.After that,we added gene D from dendritic cells targeting peptide in both ends of the KC gene respectively.We constructed recombinant lactobacillus STa-LTB-STb(K),STa-LTB-STb-co 1(KC),D-STa-LTB-STb-col(DKC)and STa-LTB-STb-col-(KCD).Western blot analysis showed that the fusion gene was expressed in Lactobacillus.Recombinant protein KCD and DKC were expressed mainly in the secreted form.To study the potential applications of protective antigen protein F4+ ETEC lactobacillus casei system as live vaccine,we chose 5-7 weeks-old BALB/c mice as experimental animals,to conduct immunological evaluation.Three times immunization program for immunization,vaccine orally the amount of(cfu = 109)live lactobacilli bacteria,PBS,pPG612.1/L.casei393,pPG612.1-KC/L.casei393 and pPG612.1-K/L.casei393 as the control.Specific IgG antibody levels of immuned mice were measured at different times.The experiments showed that the target protein are nontoxic and can induce protective immune response;recombinant protein modified by M cells and dendritic cells targeting peptide can induce higher immune response,the modified group produced the effect of higher levels of specific IgG antibodies without targeting peptides and single-targeting peptide generated significantly;M cell targeting groups can be modified by two immune protection to resist attack of virulent strain of E.coli(1 × 109CFU/only),and produced 100%protection rate.These results showed that the protective antigens constructed were safe and effective,M cells and dendritic cell targeting peptide have immune-enhancing effects.After immunization,fresh faeces,nasal fluid,external genital fluid samples of immuned mice were collected in different time periods,to determine the content of specific sIgA.Detection of Thl,Th2,Th17 expression levels by flow cytometry methods,by MTT assay to detect lymphocyte proliferation of immuned mice.The effect of oral immunization showed that immunization group PBS,pPG612.1/L.casei393,pPG612.1-KC/L.casei393 and pPG612.1-K/L.casei393,in the first 7d,feces and external genital tract can be detected higher levels of slgA antibodies,the difference was significant compared with the control group(P<0.01),M cell targeting modification group pPG612.1-KC/L.casei393 was significantly higher than unmodified group pPG612.1-K/L.casei393;after the third immunization,pPG612.1-DKC/L.casei393 and pPG612.1-KCD/L.casei393 in feces,external genital tract and nasal lavage fluid all could be detected high levels of sIgA antibodies,and after the completion of three immunization,specific sIgA content appeared the highest peak.Difference between the groups was significant(P<0.01),and also the control group(P<0.01);These results above indicate that the recombinant lactobacillus casei system can stimulate animals mucosal immune responses and systemic immune responses,and antibody levels of M cell targeting peptides and dendritic cells target peptide was significantly higher than unmodified group.The rat mesenteric immune lymph freshly isolated were diluted to 5 X 106/mL,to detect T-helper cell subsets Th1 and Th2 by flow cytometry,analysed the modifications ways of M cells and dendritic cell targeting peptide against the effect on the way of Th cell level immunomodulatory.Flow cytometry showed that the lactobacillus bacteria modified by M cells and dendritic cell targeting peptides,have undergone significant balance drift,Thl/Th2 was less than 1.Flow cytometry results showed pPG612.1-KCD/L.casei393 and pPG612.1-DKC/L.casei393 groups appeared obvious local cellular immune answer in mesenteric lymph nodes.This has the consistent results with the first 51 days,sIgA level of pPG612.1-DKC/L.casei393 is higher than pPG612.1-KCD/L.casei393.The experiment constructed recombinant lactobacillus casei with F4+ ETEC protective antigens,confirmed that M cell and dendritic cell dual targeting system strategies in lactobacillus live vector vaccines can apply effectively,and also got that pPG612.1-KCD/L.casei393 targeting strategy can produce higher immune enhancement.We determined that lactobacillus as oral vaccine has potential applications,and lay the foundation for the next step to develop enterotoxigenic Escherichia coli oral vaccine,and also provides the basic materials of practice and mechanism research to explore dual targeting antigen delivery and enhance the mucosal immune response level.