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The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×10⁶ Da to 20×10⁶ Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.

Objective: To construct the recombinant adenovirus co-expressing foot-and-mouth disease virus (FMDV) viral structural protein1-2A (VP1-2A) and porcine interferon-α (poIFN-α), and study its impact on mouse adaptive immune responses., Methods: FMDV VP1-2A genes and porcine IFN-α (poIFN-α) genes were successively cloned into pAdeno Vator-CMV5-IRES-GFP, and then both pAdeno Vator-CMV5-IRES-GFP with VP1-2A-poIFN-α and pAdeno Vator ΔE1/E3 were co-transfected into E.coli BJ5183 competent cells. The homologous recombinant plasmid rAd5VP1-2A-poIFN-α was screened and linearized by Pac I to expose the encapsidation signal and then transfected into HEK293A cells by Lipofectamine(TM)2000. The positive recombinant viruses were named rAd5VP1-2A-poIFN-α. The BALB/c mice were immunized by the recombinant adenovirus rAd5VP1-2A-poIFN-α or rAd5VP1 (adenoviral vectors solely expressing FMDV VP1) or blank adenovirus as control (Ad virus). The FMDV VP1-specific IgG, IgG1, IgG2a and cytokines IL-4, IFN-γ were determined by the ELISA, and the index of peripheral blood lymphocyte proliferation was detected by MTT assay., Results: The recombinant adenovirus co-expressing FMDV VP1-2A and porcine IFN-α (rAd5VP1-2A-poIFN-α) was successfully constructed. ELISA and MTT assay showed that compared with rAd5VP1 group, rAd5VP1-2A-poIFN-α could enhance the secretion of FMDV VP1-specific IgG, IgG1 and IgG2a, promote the secretion of IL-4 and IFN-γ, and increase the index of peripheral blood lymphocyte proliferation., Conclusion: The rAd5VP1-2A-poIFN-α could significantly improve the mouse adaptive immune responses.

Objective: To explore the biological function of the interferon stimulation reaction element (ISRE) like motif CTGAAAACGAAAGA within porcine circovirus type 2 (PCV2) Rep promoter., Methods: Two recombinant PCV2 strains, namely PCV2 1740(G-C) and PCV2 1741(A-T), were constructed by transfecting PK15 cells with site-mutated infectious clone of PCV2 strain Denta. Replication character, genetic stability and reactive character to porcine interferon alpha (poIFN-alpha) were compared among parental PCV2 and the two mutant viruses., Results: The ISRE like motif in Rep promoter was not necessary for the replication of PCV2 because two site-mutated viral genome clones both produced infectious virus. In contrast to parental PCV2, the viral antigen positive PK15 cells of the two site-mutated PCV2 were decreased. PCV2 1740(G-C) was genetically stable in the PK15 cell while PCV2 1741(A-T) was found to have another two nucleotide mutated from 1744AC1745 to 1744TT1745 between 3rd and 7th passage in the PK15 cell. After treated with 100 U/mL porcine interferon alpha, the viral antigen positive PK15 cells and virus genomes of parental PCV2 and two site-mutated viruses were both increased. But the enhancement rate of the two site-mutated PCV2 was significantly lower than parental PCV2., Conclusion: Site-mutation of ISRE like motif in Rep promoter decreased the replication and poIFN-alpha induced enhancement of PCV2 in PK15 cells. According to these above results, it maybe speculated that ISRE like motif in PCV2 Rep gene promoter contain a functional element and it may contribute to the interferon inducible enhancement of virus replication in PK15 cells.

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.

Based on the antigenic analysis of duck plague virus (DPV) gB protein, we designed a pair of primers to amplify the gene fragment encoding high antigenic domain of DPV N-terminal gB protein from the DPV genome. The cloned gene was digested with EcoR I and Hind III and then inserted into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level after induced with IPTG. The expressed product was analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) existed as inclusion body, which was about 42.4kDa and showed specific immunoreactivity with anti-DPV sera. The recombinant gB1 protein was purified with His-Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against DPV with the purified gB1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 6.5 microg/mL and the optimal dilution of serum was 1 : 80. The positive criterion of this ELISA assay was OD(the tested serum) > 0.4 and OD(the tested serum)/OD(the negative serum) > 2.0. The ELISA was done on 700 sera that were preserved in Shandong, Jiangsu Provinces, and were detected by igB1-ELISA and iDPV-ELISA with duck plague virus as the coating antigen respectively. The agreement ratio between the two methods was 95.6%.