Isolation And Identification Of Porcine-originated Escherichia Coli Nissle 1917 And Its Initial Assessment As An Oral Vaccine Vector

The probiotic Escherichia coli Nissle 1917 strain was first isolated from the health soldier's small intestine by Alfred Nissle in 1917 when the diarrhea was endemic . Its serotype formula is O6:K5:H1.It can locate in the host intestines and keep the intestinal microflora homeostasis. It also plays a significance role in the intestinal mucosal immunization as it's able to stimulate the intestinal immune system to produce immunoglobulins and cytokines. This strain has been well characterized, in particular for its probiotic properties in humans, mice and cattle, and has been licensed for use in medical applications in humans and cattle. The probiotic and safety properties for use in pigs remain to be validated, and the application of E. coli Nissle 1917 is therefore not yet permitted for this species. Now, there is no related report in China.Bacterial vector-based vaccine is constructed by inserting the DNA fragments which encode some specific antigens from the pathogens or the target antigens into the attenuated bacteria or symbiotic bacteria as live vehicles, to express, deliver and present the encoded antigen and effectively stimulate immune response of the host cells as the attenuated bacteria or symbiotic bacteria colonize, multiplicate and grow in the host. Bacterial vector-based vaccine with capability of producing both humoral and cellular immunue responses, especially mucosal immunue response can prevent one or multiple specific pathgen infection as we expect to. It is of note that the attenuated bacteria as antigen carrier have a certain risk of virulence revert, the study of probiotics to be used as protein, antigen delivery vehicles becomes a new highlight. With the development of the protein expression technique for probiotics, The Escherichia coli Nissle 1917 strain has a variety of properties which make them attractive candidates for oral vaccination purposes.The target of this study is to verify the presence of Escherichia coli Nissle 1917 as a natural swine isolate and isolate E. coli Nissle 1917 from the intestinal microflora of different pig population. We explored colonization properties of Escherichia.coli Nissle 1917 in mice via the peroral recombinant bacteria-GFP expressing green fluorescent protein(GFP) and immune response after oral vaccination of mice with recombinant Escherichia coli Nissle 1917-fedF expressing F18 fimbrial adhesin fedF.Gabriele desined five pairs of PCR primers which are based on the chromosomally encoded major fimbrial subunit genes fimA (type 1 fimbriae) and focA (F1C fimbriae), and the two small cryptic plasmids pMUT1 and pMUT2 to detect human-originated Escherichia coli 1917. Kleta demonstrated that Escherichia coli 1917 could be detected with the five PCR primers in DNA templates prepared from the pig feaces. The human-originated Escherichia coli 1917 was chosed as the positive one in our study.We tested 135 pieces of fresh piglets faeces in a collection of different breeds and ages by PCR with five pairs of primers.The result showed that the expected five specific target fragments including target fragment 427bp of pMUT2(a) were amplified from two pieces of the DNA samples and porcine-originated E. coli Nissle 1917 was initially confirmed in the intestine of pigs. pMUT2(a) as a DNA probe fragment was purified, ligated to T vector and then recombinant plasmid was amplified.The purfied target fragment was made into probe using non-radioactive digoxigenin random primer labeling. The pMUT2(a)probe had a high sensitivity of 16pg. Two positive strains were srceened by the method of in situ hybridization from the 2 positive samples respectively. Finally, positive clones were further confirmed by combination of serological test, PCR and sequencing.Recombinant bacteria Nissle 1917-GFP can express green fluorescent protein and Nissle 1917-fedF which was cultured under anaerobic conditions can express subunit fedF of the Escherichia coli adhesin F18. The recombinant bacteria was orally administrated to the ICR mouse. The retention time of the recombinant bacteria and IgG serum titres against fedF were tested separately. The results showed that Nissle 1917-fedF stained 24 hours longer than Nissle 1917-GFP in the mouse. The results showed that IgG serum titres against fedF raised as early as 14 days and increased significantly at 33 days. While there was no effect in Nissle 1917 and Nissle 1917-GFP groups. Our research of oral vaccine candidate of Nissle 1917 provided a new way and foundation for the prevention and treatment of diarrhea disease.