Ge JunWei

High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.

Expression of infectious pancreatic necrosis virus (IPNV) VP2–VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

High-level prokaryotic expression of envelope exterior of membrane protein of porcine epidemic diarrhea virus

Abstract The truncated fragment M′ gene, encoding the exterior of the viral envelope protein of PEDV, was subcloned into prokaryotic expression vector pGEX-6p-1. The recombinant plasmid pGEX-6p-M′ was constructed and transformed into E. coli BL21(DE3)pLysS for expression. SDS-PAGE analysis showed recombinant truncated M′ protein was highly expressed by pGEX-6p-M′ and the product fusion protein GST-M′ reached 45% in the total bacteria proteins with the analysis of software AlphaImager2200. The preliminary purified recombinant protein was evaluated for its antigenicity and reactivity through Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody against M protein of PEDV and porcine polyclonal anti-PEDV antiserum as the primary antibody. The results indicated the recombinant truncated M′ protein should be candidate as a feasible recombinant diagnostic reagent.

Prokaryotic expression of VP3 gene of infectious pancreas necrosis virus and antigenicity of expressed product

Infectious pancreatic necrosis(IPN) virus,the etiologic agent of infectious pancreatic necrosis in salmonid fish,causes significant losses to the aquaculture industry.The gene for the viral inner capsid protein(VP3) was amplified by RT-PCR method from IPNV,and cloned into pET30b vector.The expression of recombinant plasmid pET30b-VP3 in E.coli BL21(DE3) was induced and detected by SDS-PAGE analysis.The predicted molecular weight for unmodified r-trunc VP3 was approximately 30 ku and this was found to be the case for E.coli protein.The amount of expression made up 30 percent of the bacteria protein total expression by thin layer scanning analysis.The results showed that the VP3 gene of IPNV can express successfully in E.coli BL21.The fusion protein was purified with ProBondTM resin from the suspension centrifuged and the antisera against VP3 protein was produced.The pET30b-VP3 fusion protein can be recognized by the positive serum of IPNV by Western-blotting analysis.The prepared antisera reacted specifically with IPNV antigen by indirect ELISA.The antisera against VP3 protein had OD values at least twice that obtained for the negative control serum at a dilution of 1 :25 600.The results showed that the expressed VP3 protein was immunogenical and antigenical which is the same as the natural IPNV VP3 protein.In this experiment the IPNV VP3 protein was expressed successfully by using prokaryotic expression system.The expressed fusion protein was active and the antisera against VP3 protein were produced.