C3d-M28 enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus / 生物工程学报

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.

Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-gamma and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.

Diagonsis establishment of fluorescen quantitative PCR assay for pseudorabies wild-type virus and vaccine virus / 生物工程学报

We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.

Expression of truncated gE gene of pseudorabies virus (PRV) and primary application in differential diagnosis of PRV vaccination and infection / 生物工程学报

With the application of gE gene deleted pseudarabies virus (PRV) vaccine worldwide, a corresponding differential diagnosis based on gE glycoprotein is needed in the project of PRV eradication. In this study, PRV gE gene without signal and transmembrane region was amplified by PCR and cloned into pGEX-6P-1, generated pGEX-gE. After transformation of BL21 with pGEX-gE, an expressed fusion protein(about 63kD) was identified by SDS-PAGE. The recombinant proteins are produced as inclusion bodies. By changing the inductive conditions, the formation of inclusion bodies was inhibited and tended to increase the percentage of soluble recombinant protein. The antigenic reactivity of the recombinant protein was confirmed by Western blotting with polyclonal antibodies against PRV. Using purified recombinant tgE as antigen, an ELISA was developed to detect sera of PRV infected pigs and sera of pigs immunized with gE-deleted PRV vaccine. The total of 400 serum samples collected from field were comparatively tested with the tgE-ELISA and a commercial competitive ELISA based on monoclonal antibody against gE, the results indicated that the coincidental rate between the two tests is about 94%.

Construction and characterization of a pseudorabies virus TK-/gG- mutant / 生物工程学报

To construct a TK-/gG- mutant of pseudorabies virus, the gG-detected transfer vector pUSKKBB and genomic DNA of pseudorabies virus TK-/gG-/LacZ+ were co-transfected into IBRS-2 cells. Transfection progeny were plated onto PK-15 cells and incubated for 2 days under methylcellulose. Then the overlay was removed and replaced by 1% low melting point agarose in DMEM supplemented with 150 microg/mL X-gal. After 2 days, white plaques were screened for and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confirmed. Sequence of the PCR product revealed that there were 1,176 bp detection in gG gene of the PRV TK-/gG- mutant. Amplifying the gG-deleted gene of different generations of the TK-/gG- mutant showed that the mutant was stable within PK-15 cells. TCID50 assay indicated that the recombinant virus grows well on PK-15 cells. The mice immunized with the TK-/gG- virus showed no sign of abnormality. As a control, all mice inoculated with PRV strain died from the infection. All mice that received TK-/gG- survived after a lethal PRV challenge. However none of the mice injected with phosphate-buffer saline (PBS) survived from the challenge. The above results demonstrated that the recombinant virus could be a candidate marker vaccine strain for eradicating pseudorabies in pig herds.