Immune Responses Of Murine Cells Stimulated By Flagellin Of Salmonella Typhimurium And Its Adjuvant Effects

Bacterial flagellin has unique structure and function with strong antigenicity(antigen H).It helps bacteria invade in cells.Flagellin can stimulate cell producing proinflammatory cytokine, and it plays a important role in combined innate immune with adaptive immune.Newcastle disease(ND) is one of the major infectious diseases suffered in the poultry industry,the Office International des Epizooties(OIE) list it as the statutory notification of disease.It has been reported that fusion protein located in NDV envelope is involved in viral pathogenicity, including virus penetration and cell fusion.In addition,it is the major protective antigen with good immunogenicity.Using the eukaryotic expression plasmid pVAX1-F containing the complete gene of fusion protein of NDV strain F48E8 as the template,a 594 bp partial fragment was amplified by PCR. After cloning and sequencing,the recombinant prokaryotic expression plasmid pGEX-6P-1-F was constructed,and then plasmid transformed into expression host BL21.The positive recombinant strain was renamed BL21(pGEX-6P-1-F).The result of SDS-PAGE showed that fusion protein was expressed effectively induced by IPTG,the amount of fusion protein takes 23%in total bacterial expression proteins.The result of Western blot showed there is a 46 KD specific band.Meanwhile the flagellin of Salmonella typhimurium SL7207 was extracted from its M-broth culture.The results of SDP-PAGE and Western blot confirmed that the putified flagellin was 51 KD protein.The peritoneal marcophages of C57BL/6 mice were harvested and cultured with 0.5μg/ml or 1μg/ml endotoxin-free flagellin in 96 well cell culture plates for 2,4 or 6 hours.The cells were harvested at the indicated times poststimulation for total RNA extraction.Total RNA was reversly transcribed into cDNA and then amplified forβ-actin,IL-1β,TNF-α,IL-6 by RT-PCR. PCR products were electrophoresed in a 1.5%agarose gel.The result showed that flagellin can initiate cellular immune responses with 0.5μg/ml within 2 h.Furthermore,the macrophages were stimulated with 0.5μg/ml flagellin,fusion proteins,flagellin mixed fusion proteins for 4 h respectively.The result indicated that flagellin and flagellin mixed with fusion proteins can trigger strong proinflammatory response,and fusion proteins only can stimulate weak proinflammatory response.In order to analyze the phenotype changes,the macrophages were cultured with contentration 10μg/ml flagellin for 2 or 10 h.The FACS results showed that CD80,CD86 on the surface of macrophages markedly increased post stimulation 10 hours compared with those stimulated for 2 hours.BALB/c mice were immunized i.p.with 10μg flagellin for 12 or 24 hours.Low-density cells(LDC) from spleen were isolated.Cells were cultured with contentration 10μg/ml flagellin for 12 or 24 h.For LDC analysis,cells were incubated on ice with CD11c-FITC/CD40-biotin, CD11c-FITC/CD80-biotin,CD11c-FITC/CD86-biotin.FACS data showed the expression levels of CD40,CD80 on LDC surface were increased when cells were cultured with flagellin for 12 or 24 hours,and their levels in 12 hours were higher than those in 24 hours.In contrast,the levels of CD80 was not obviously changed.C3H/HeJ mice were immunized s.c.with flagellin,fusion proteins,flagella mixed with fusion proteins or fusion protein with Freund's adjuvant respectively.Injection dose was 10 micrograms flagellin or/and 80 micrograms fusion protein per mouse.After the third immumization,the titer of antibody against fusion protein in mice immunized with fusion proteins mixed flagellin was reached to 4266,while the level of antibody in mice immunized with fusion proteins mixed with Freund's adjuvant was up to 7680,and only 213 in mice immunized with fusion proteins.IFN-γand IL-4 secreting cells in C3H/HeJ mice splenocytes were detected by ELISPOT assay.The results showed that fusion protein mixed flagellin induced mice produced higher IFN-γsecreting cells.Meanwhile it also produced secreting IL-4 cells. Fusion protein mixed with adjuvant induced mice generated a banlance responses of secreting IFN-γor IL-4 cells.