| Enterotoxigenic Escherichia coli (ETEC) is an important causative agent of diarrhea in neonatal and weaned piglets, and the high morbidity and mortality of this disease results in severe economic losses. F4+(K88) ETEC is the most prevalent strain causing piglet diarrhea. Major subunit of the F4 fimbriae, Fae G, possesses adhesive and antigenic properties and is currently viewed as a prospective vaccine candidate. Lactococcus lactis (L. lactis) is generally regarded as safe (GRAS), and is a promising antigen delivery vehicle for mucosal immunization with many special properties other than some vehicles. To safely and effectively deliver F4 fimbrial adhesin FaeG to the immune system, recombinant L. lactis expressing FaeG was constructed. Immunogenicity of recombinant FaeG (rFaeG) was detected and the effect of immune responses in mice and piglets immunized with recombinant L. lactis were evaluated.The construction of FaeG expressing recombinant L. lactis The truncated faeG gene was amplified by PCR from plasmid DNA of ETEC C83907 (O149:K91, K88ac) and cloned into pNZ8148, a vector for cytoplasmic expression, and pNZ8112, a vector for secretory expression, then they were transformed into L. lactis NZ9000 respectively. L. lactis NZ9000 (pNZ8148-faeG) and L. lactis NZ9000 (pNZ8112-faeG) were constructed. After two recombinant L. lactis were induced with 5 ng/ml of nisin, FaeG were expressed by two recombinant L. lactis. But FaeG was produced with intracellular form in L. lactis NZ9000 (pNZ8112-faeG) and not in culture supernatants. A protein bind with MW about 27.0 kD was detected by SDS-PAGE, and the products of rFaeG were added up to 10% and 13% of soluble whole-cell proteins in L. lactis NZ9000 (pNZ8148-faeG) and L. lactis NZ9000 (pNZ8112-faeG) respectively. Western blot analysis indicated that rFaeG possessed good reactionogenicity.The study on immunogenicity of FaeG expressed in recombinant L. lactis for mice BALB/c mice were used as a model to evaluate the immunogenicity of recombinant L. lactis. 112 mice were randomly divided into 14 groups and immunized with L. lactis NZ9000 (pNZ8148-faeG) and L. lactis NZ9000 (pNZ8112-faeG) via subcutaneous and oral routes respectively. The results suggested that subcutaneous immunization in mice with L. lactis NZ9000 (pNZ8148-faeG) induced a significant increase IgG titer (maximum,11.32 log2 titer) in the serum and the number of IgG antibody-secreting cells (ASC,817/107 MC) in the spleen (SP), which indicated that recombinant L. lactis induced F4-specific systemic immune response via subcutaneous vaccination. Oral immunization of mice with L. lactis NZ9000 (pNZ8148-faeG) induced F4-specific serum IgG and fecal IgA which reached up to 9.65 and 6.32 (log2 titers) respectively, and enhanced the number of ASC in the SP, mesenteric lymph nodes (MLN), and Peyer's patches (PP) by ELISPOT assay. These indicated that recombinant L. lactis induced F4-specific mucosal and systemic immune responses via oral route. Furthermore, a high dose and CTB were set to study whether the dose and mucosal adjuvant could affect the immune responses via oral route. Results suggested that oral dose of recombinant L. lactis and mucosal adjuvant of CTB could be important factors which improved the F4-specific immune responses. Similarly immune responses were induced in mice by immunization with L. lactis NZ9000 (pNZ8112-faeG).The study on immunogenicity of FaeG expressed in recombinant L. lactis for weaned piglets via intramuscular route To further determine immune responses of the rFaeG expressed in L. lactis for piglets,28 six week old weaned piglets were randomly divided into 4 groups and immunized L. lactis NZ9000 (pNZ8148-faeG) via intramuscular route. Results showed that recombinant L. lactis induced F4-specific IgG coupled with low levels of IgA and IgM in serum. The levels of F4-specific serum IgG in piglets immunized with recombinant L. lactis were significantly higher than those in the control group at 21,28, 35 and 42 dppi (P<0.05), and serum IgA at 21 and 28 dppi (P＜0.05), F4-specific IgA and IgM antibodies in the jejunum and ileum were found in piglets, and the levels of them were higher than those in the control group (P>0.05), but there was no significant difference between the groups. Furthermore, ASC of IgA and IgM induced in the spleen, duodenum, jejunum, ileum and mesenteric lymph node by immunohistochemisty assay were higher than those in the control group. These results indicated that intramuscular vaccination of weaned piglets with recombinant L. lactis induced systemic immune response and a weak mucosal immune response.The study on immunogenicity of FaeG expressed in recombinant L. lactis for weaned piglets via oral route Immune responses induced in weaned piglets following oral administration of L. lactis NZ9000 (pNZ8148-faeG) were studied.33 six week old weaned piglets (du chang da species) were randomly divided into 5 groups and immunized with L. lactis NZ9000 (pNZ8148-faeG) via the oral route, the immunization induced F4-specific serum IgG, IgM and IgA responses, and the levels of F4-specific serum IgG and IgA were significantly higher than those in the control group at 28 dppi (P<0.05). The level of IgA in the jejunum was significantly higher than that in the control group (P＜0.05). ASC of IgA, IgM and IgG were also induced in the spleen, duodenum, jejunum, ileum and mesenteric lymph node, ASCs of them were higher than those in the control group by immunohistochemisty assay, and IOD of IgM in the jejunum was significantly higher than that in the control group (P<0.05). Results indicated that oral immunization of weaned piglets with recombinant L. lactis was able to induce both systemic and mucosal immune responses.The present study constructed successfully recombinant L. lactis expressing FaeG, the immune responses in mice and piglets induced with recombinant L. lactis indicated that recombinant FaeG possessed good antigenicity. The present study is the first report of oral and intramuscular immunization with recombinant antigen expressed in L. lactis to evaluate the mucosal and systemic immune responses, and set a new sight in researching the vaccine for protection of diarrhea in neonatal and weaned piglets using L. lactis as an antigen vector, and was also provide an annotation of developing oral live vector vaccine with L. lactis for piglets use, promoting the growth of animal health. |
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