| Newcastle disease(ND) is an acute and highly contagious disease caused by Newcastle disease virus(NDV).The morbility and mortality is very high in China even in the world wide, so it is very harmful to the poultry industry and cause enormous economic loss. The surfaces of NDV particles contain two important functional glycoproteins:the fusion(F) and hemagglutinin-neuraminidase(HN) proteins. The HN protein of NDV is a multifunctional protein, which is recognizes sialic acid-containing receptors on cell surfaces.The F protein promoting the fusion activity mediates both virus-cell and cell-cell fusion. In this study, Lactobacillus casei (L.casei) was selected as vectoer strain to express HN gene, which combined physiological function with specific immunity together. The experimental aim is to establish an antigen delivery system for foreign antigen HN of NDV in order to protect chicken from NDV infection.In this study, HN gene of NDV was cloned into expressing vector with Amp and Chl double labellings, which can shuttle and express in E.coli and L.casei. We have developed a surface antigen display system using pgsA (poly-γ-glutamate synthetase A) as an anchoring matrix. The recombinant fusion proteins comprising of pgsA and fimbriae protein of HN were stably expressed on L.casei. The recombinant vector pLA-HN was transformed into the competent cells L.casei. Western blotting analysis with chicken-anti-NDV polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 100kDa. The HN fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. In addition, Chickens of 20-day-old were orally inoculated with the pLA-HN/L.casei. At 1.5 to 108h post administration, the animals were sacrificed. The stomachal and intestinal contents were cultured and counted on MRS plates. Three hours after ingestion, the pLA-HN/L.casei was detectable in all samples. At 48h post administration, the microorganism was present in the intestine (stomach, jejunum, ileum, and caecum) with maximum concentrations.The recombinant lactobacilli (1×104CFU/g) were still persisting in the GI tract At 6h to 84h post administration. The results showed that the pLA-HN/L. casei. could plant the chicken's gastrointestinal tract.Oral and intranasal immunization of chickens was performed with the recombinant strain L.casei harboring pLA-HN or LaSota vaccine. Specific anti-NDV IgG in the serum and specific anti-NDV secret immunoglobulin A (slgA) antibody in the lung, intestines fluid and feces of chickens were detected by indirect ELISA and HA-HI. Five weeks after immunization with the recombinant L.casei or LaSota vaccine, The chickens orally or intranasally immunized with pLA-HN/L.casei and LaSota vaccine were challenged with standard-type NDV(F48E8). The results showed that special serum IgG and slgA antibodies were detected in the vaccinated group. One handred pesent and 88% chichkens survived in oral and intranasal LaSota immunization groups whereas 72.8% and 63.7% chichkens survived in oral and intranasal recombinant pLA-HN/L.casei immunization group after challenged with F48E8, while all of the chickens dead in the control group. These results indicated that the recombinant pLA-HN/L.casei we prepared in this study can induce immune response in immunized chickens and protect chickens from NDV F48E8 challenge.
|
PDF Full Text Request