Kuoshi Jin

Expression of AIV Subtype H5HA, H7HA and H9HA Hemagglutinin Gene in Pichia pastoris

The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.

Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing ORF2/ORF1 of PCV2 and P1 2A of FMDV

A recombinant fowlpox virus (rFPV-ORF2ORF1-P12A) containing the open reading frame ORF2)/ORF1 DNAs of the porcine circovirus 2 (PCV2) (strain, Inner Mongolia) and foot-and-mouth disease virus (FMDV) capsid polypeptide of O/NY00 was evaluated for its abilities to induce humoral and cellular responses in piglets. In addition, we examined its abilities to protect cell cultures against a homologous virus challenge. To approach the feasibility of different united ways of immunization, the recombinant fowlpox virus rFPV-ORF2ORF1-P12A and the recombinant DNA plasmid pVAX1-IL18-ORF2ORF1 were used to immunize the pigs in “prime-boost”programme. We observed that priming the pigs with DNA plasmid pVAX1-IL18-ORF2-ORF1, followed by boosting with the recombinant virus rFPV-ORF2ORF1- P12A produced partially cellular immunity and humoral immunity. Control groups were inoculated with wild-type fowlpox virus (wtFPV) and phosphate buffer saline (PBS). All animals vaccinated with rFPVORF2ORF1- P12A developed specific anti-PCV2/anti-FMDV enzyme-linked immunosorbent assay (ELISA) and neutralizing antibodies and also showed T lymphocyte proliferation response. The antibody level produced by PCV2 was lower than that of O type FMDV to 1:20 and 1:200 respectively. We examined specific cytotoxic T lymphocyte (CTL) production in pigs serum and T lymphocytes (CD4, CD8, and CD4/CD8 double positive T cells) in the peripheral blood. First inoculating pVAX1-IL18- ORF2ORF1 and then rFPV-ORF2ORF1-P12A, had considerably higher CD4+, CD8+ and CD4+CD8+ T lymphocytes subgroups compared with the control groups. Whether the ratio between effective cells and target cells was 50:1 or 25:1, the specific CTL of experimental groups had much more significant differences with the control (FPV), even still the group of priming nucleic acid vaccine boosting recombinant virus had the bravest cytotoxicity of specific CTL. Moreover, the E/T ratio of 50:1 was more excellent. Following infection respectively with a mixture of a pathogenic strain of PCV2 (strain, Inner Mongolia)/FMDV (O/NY00) and neutralizing antibody, PK15 cells (BHK21) inoculated with recombinant fowlpox virus (rFPV) showed less (P < 0.05) yellow-green fluorescence and cytopathogenesis, suggesting the establishment of partial protection against PCV2/FMDV infection. The results show that the immunization programme here, in which pVAX1-IL18-ORF2ORF1 DNA vaccine was inoculated firstly and rFPV-ORF2ORF1-P12A was followed, is viable and indicates the potential use of a fowlpox virusbased recombinant vaccine for the control and prevention of PCV2/FMDV infections. Key words: PCV2, rFPV, FMDV, immune response, prime-boost.

Molecular Design and Immunogenicity of a Multiple-epitope FMDV Antigen and DNA Vaccination

This article reports the design and construction of a multiple-epitope foot and mouth disease virus (FMDV) antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-γ and cytotoxic activity from mice immunized with pA were significantly increased. pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asia1 FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.