| Enterotoxigenic Escherichia coli (ETEC) strains that produce K88 (F4) fimbriae on their surfaces commonly induce diarrhea in piglets, which is an important cause of both mortality and reduced growth rate resulting in heavy economic losses. With the F4 fimbriae, the bacteria adhere to the F4 receptor (F4R) on small intestinal villi of piglets, then colonize the small intestine and produce enterotoxins which cause electrolyte imbalance in the gut, inducing diarrhea, eventually leading to death. K88 (F4) fimbriae are the best-characterized adhesins composed mainly of several hundreds of identical adhesive subunits called FaeG, as well as some minor subunits. As a result, the research of immune against colibacillus diarrhea of newborn piglet has been given much attention in the present situation. In this study, a recombinant L. lactis producing FaeG extracellularly was constructed and orally administered to ICR mice. Immunized mice were then challenged with ETEC to determine the level of potential protective response induced by recombinant L. lactis. This will provide a new way to counter ETEC infection in animals. The main research contents and results were as follows:1. Secretory Expression of K88 (F4) fimbrial adhesin FaeG in Lactococcus lactis NZ9800The about 800 bp fragment encoding faeG was cloned from the Enterotoxigenic Escherichia coli C83549 genome with a pair of primers designed according to the sequence of faeG in GenBank. Strong homology of the nucleotide sequence and amino acid sequence of the faeG gene was found between ETEC C83549 and other ETEC sources. The nucleotide sequence identidies were 98%-100%; the amino acid sequence identidy was 99%. After digested with BamHI/XbaI, the gene was inserted into spNZ8048 vector and recombinant vector spNZ8048:faeG was obtained. spNZ8048-faeG was then transformed into L. lactis NZ9800. FaeG expression in L. lactis [spNZ8048-faeG] was induced with nisin. Expression of recombinant protein FaeG was analyzed by SDS-PAGE and Western blot. Results showed that the FaeG protein expressed in-cellular was about 26.0 kDa, and it could be secreted into culture medium successfully after cleaved with signal peptide Usp45, at the size of about 25 kDa, which was consistent with the theoretical Mw of FaeG (23.5 kDa).2. The immunoprotection effect of the recombinant L. lactis on ICR mice challenged with ETEC C83549 ICR mice (Mus musculus) were randomly divided into four groups, which was supplicated (immuned) with PBS, L.lactis[spNZ8048], L.lactis[spNZ8048-faeG] and L.lactis [spNZ8048-faeG]+L.acidophilus respectively. All mice were immuned once twice weeks, with total three times and three days for each immunation. Serum IgG level was detected by ELISA a week after each immunation. On the 14th day after last immunization,10 mice were challenged with 5.34×109 cfu/ml of live ETEC C83549 (100LD50). The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C83549 challenge were evaluated in ICR mice. The results showed that serum IgG was found in groups immuned with L.lactis[spNZ8048-faeG]+L.acidophilus and L.lactis[spNZ8048-faeG] on the 10th day after first immunation, and the IgG level was increasing with time, with a maximum level reached on the 35th day. The death rates was respectively 100%,80%, 10%and 0%for PBS, L.lactis[spNZ8048], L.lactis[spNZ8048-faeG]和L.lactis [spNZ8048-faeG]+L.acidophilus groups. The relative percent survival (RPS) of groups L.lactis[spNZ 8048-faeG] and L.lactis [spNZ8048-faeG]+L. acidophilus reached 88.9% and 100%respectively. These results indicated that oral immunization of L. lactis [spNZ8048-faeG] could induce an immune response protective upon challenge with live ETEC in ICR mice.3. Effect of immunical function on immuniated mice of the recombinant L. lactisTo elucidate the mechanism of immunation protection effect of FaeG recombinant L. lactis, immune indices including thymus index, lymphocyte cell proliferation, sIgA level and the number of plasma cell producing IgA in mucous membrane, CD4+ ratio, CD8+ratio, CD4+/CD8+ratio, serum IgG, IgM, IgA, IL-2 and IL-10 levels were detected. The results showed that FaeG recombinant L. lactis could improve the development of mice immune organ, increase the proliferation ability of lymphocyte cells, incentive the gut mucosal immunity, increasing the secretion level of sIgA, significantly increase the levels of IgG, IgM, IgA, IL-2 and IL-10, but no significant effect was found in peripheral blood T-lymphocyte subpopulations. Also, oral administration of L.acidophilu could enhance the immune responses.4. Effect of the recombinant L. lactis on intestinal microflora of ICR miceThe effects of the recombinant L. lactis [spNZ8048-faeG] on intestinal microflora, microvilli density and growth performance of ICR mice were studied. Results showed that though no significant difference was found in mice growth between the control and immuned groups, L. lactis [spNZ8048-faeG] could significantly increase the microvilli density of ICR mice and the highest microvilli density was found in L.lactis[spNZ8048-faeG]+L.acidophilus group. In comparison with the two control groups, the numbers of Lactobacillus and Bifidobacterium in groups L.lactis[spNZ8048-faeG] and L.lactis [spNZ8048-faeG]+L.acidophilus were increased (P>0.05), while the numbers of Enterobacter (P>0.05) and Enterococcus were decreased (P<0.05). The results suggested that recombinant L. lactis [spNZ8048-faeG] expressing FaeG effectively protect ICR mice against ETEC C83549 challenge.
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