| The flagellum is the motility device of Salmonella, and flagellin is the subunit of its filament. Flagellins have been investigated as protective antigens for vaccination against Salmonella spp. which at the same time are well known as the ligands of Toll-like receptor 5 (TLR5) and involved in innate immune responses during Salmonella infection. Previous researches have shown that these innate immune responses induced by flagellins will help to elicit antigen-specific adaptive immune responses, indicating the potential adjuvant activity of flagellins.Avian influenza (AI), officially notified by OIE, is a contagious disease of poultry caused by influenza A virus, which is part of the Orthomyxoviridae family of flu viruses. In recent years, however, some multiple avian influenza A subtypes including H5N1 have been discovered to infect humans and cause human diseases, which has attracted wide attentions from medical and veterinary fields from different countries. Matrix protein 2 (M2), the third largest transmembrane protein expressed on the surface of AIV, is highly conserved in various subtypes, and its major antigenic determinant is localized in M2e, the extracelluar of M2.Newcastle disease (ND), another notified disease by OIE, has a notable effect on domestic poultry due to its high susceptibility and the potential for severe imparts of an epidemic on the poultry industries. Vaccination is one of the main measures to reduce the likelihood of outbreaks. The fusion protein (F) which is one of the transmenbrane glycoproteins of Newcastle disease virus (NDV) is directly responsible for virus penetration and cell-cell fusion and associated with viral pathogenicity. However, it's also reported as a good immunogen, serving as the key protective antigen against NDV. In this study, we focused on the adjuvant effect of flagellin, constructed chimeric flagellin fliC/M2e2 and fliC-F fusion protein respectively, and analyzed their immunogenicity.1. Flagellin as a vector to deliver Avian influenza virus M2e and its immunogenicity analysisUsing pET-fliC as the template, the upstream of fliC gene named as fliC-AC was amplified by PCR with primers fliC-A and fliC-C. Similarly, the downstream of fliC named as fliC-DB was cloned using primers fliC-D and fliC-B. A construct encoding two tandem copies of M2e (aa 2-24) derived from the consensus sequence of H5N1 subtype AIV was chemically synthesized as a DNA concatemer. Then by overlap PCR, fliC-AC-M2e2 fragment was amplified by using primers fliC-A and M2-F with the templates fliC-AC and M2e2 genes. Finally, to achieve the final goal of inserting M2e2 gene into the hypervariable region of fliC, use fliC-AC-M2e2 and fliC-DB as the templates, fliC-A and fliC-B as primers to amplify fliC/M2e2 chimeric gene. Then the chimeric gene was inserted into pET30a+ and the recombinant plasmid was named as pET-fliC/M2e2.The recombinant plasmid pET-fliC/M2e2 was then electro-transformed into the strain LB5000 of Salmonella typhimurium and the recombinant bacteria was named as LB5000(fliC/M2e2). Then the transduction was conducted between LB5000 (fliC/M2e2) and Salmonella dublin strain SL5928 using bacteriophage P22HT int. The positive transductants were further identified by PCR, with the whole genome of transductants as the template to amplify fliC/M2e2 gene and M2e2 gene. These positive transductants were further analyzed by motility observation in semisolid medium and under transmission electron microscopy, and followed by biochemical and serological identification. FliC/M2e2 protein was then extracted from the positive transductants to identify by Western blot, using murine polyclonal anti-fliC antibodies as primary antibody. Finally, the positive transductant was named as SL5928(fliC/M2e2).The recombinant chimeric protein fliC/M2e2 was extracted from SL5928(fliC/M2e2). C3H/HeJ mice were immunized subcutaneously (s.c.) with 50μg of fliC/M2e2. M2e2-specific IgG antibodies were detected by ELISA, and the results were analyzed with SPSS15.0. The results showed that at the 14th day post boosting, there was a significant difference between fliC/M2e2-immunized group and blank control group (P <0.05), while negtive results were gained in fliC-immunized group and M2e-immunized group (P >0.05). It demonstrated that M2e2 gene, inserted into the hypervariable region of flagellin, haven't impacted the expression of chimeric flagellins in Salmonella dublin and the chimeric flagellin could be used as a adjuvant to enhance the induction of M2e-specific immunity.2. Expression of fliC-F fusion protein and its immunogenicity analysis in miceThe flagellin encoding gene fliC of Salmonella typhimurium was amplified by PCR, using pET-fliC as the template with primers FF1 and FF2. And the F gene encoding partial epitope of F protein (aa 147-344) was also cloned by PCR, using pVAX1-F as the template with primers FF3 and FF4. The two genes were tandemly ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)2 and the fusion gene was inserted into pET30a+ to construct the recombinant prokaryotic expression plasmid pET-fliC-F.Then the recombinant plasmid was transformed into E.coli BL21(DE3) and induced to express the desired fliC-F fusion protein. Western blot analysis showed that the fliC-F fusion protein could react with murine antisera against fliC or F protein respectively. It indicated that both of the two elements of fliC-F fusion protein could keep their own natural conformations. C3H/HeJ mice were immunized with 100μg of fliC-F via s.c. route. As described above, F-specific IgG antibody was detected by ELISA, and the results were analyzed with SPSS15.0. The results showed that at the 14th and 21th day post boosting, there was a significant difference of serum antibody titers between fliC-F-immunized group and blank control group (P <0.05). And there was also a significant titer difference between oil emulsified vaccine-immunized group and blank control group (P <0.05) at the 14th day after post boosting. Compared with blank control group, F-immunized group and fliC-immunized group displayed no obvious differences (P >0.05) in all corresponding periods. It demonstrated that fliC-F fusion protein expressed by E.coli could elicit strong F-specific serum antibodies in the absence of supplemental adjuvants.
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