Immunosuppression mediated by porcine FcγRⅢin PRRSV infection to PAMS / 中国免疫学杂志

Objective:To study immunosuppression mediated by the porcine FcγRⅢ in porcine reproductive and respiratory syndrome virus ( PRRSV ) infection to pulmonary alveolar macrophages ( PAMS ).Methods: In this study pulmonary alveolar macrophages cells were treated with containing 200 TCID50 PRRSV,lipopolysaccharide (LPS) (100 ng/ml) and purified mouse anti-pig FcγRⅢIgG (550 μg/ml) separately,simultaneously,PAM cells treated with purified mouse anti-pig FcγRⅢIgG (550 μg/ml) was infected by 200 TCID50 PRRSV ,untreated PAM cells as the control group.Each group were post-cultured 12,24,36,48,60,72 h, the cells and the supernatant were collected.The dynamic variation of PRRSV RNA copies in inoculation group were detected by using real-time fluorescence quantitative PCR method.mRNA level of IFN-αand TNF-αin each group were detected by using relative fluorescence quantitative PCR.Results:The result showed that mRNA level of IFN-αwas improved during PRRSV infection to PAMS 12-24 h,and mRNA level of IFN-αwas inhibited during 36-72 h,then mRNA level of IFN-αrecovered normally; mRNA level of TNF-αwas increased slightly post-infection 12-72 h.IFN-αand TNF-αmRNA levels of PAM cells treated with LPS were both up-regu-lated,using the purified mouse anti-pig FcγRⅢ IgG to treat the PAM cells,selective activation of porcine FcγRⅢ in the PAM cells down-regulated significantly mRNA levels of IFN-αand TNF-α.PRRSV infection assay mediated by selective activation FcγRⅢof the PAM cells inhibited antiviral cytokine ( IFN-αand TNF-α) mRNA levels.Conclusion:The results show selective activation of FcγRⅢinhibited significantly mRNA levels of the antiviral cytokine IFN-αand TNF-αof host cells,and innate antiviral immune response to PRRSV infection.

Expression of porcine interleukin-18 in baculovirus/insect cells / 生物工程学报

IL-18, as a polyphonic cytokine, is important in immune response and physiologic function. We designed one pair of primers, amplified the porcine IL-18 gene fused with a C-terminal 6xHistidine tag, and then subcloned into the pFastBacDual of Baculovirus transfer vector and transformed into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into insect cells, the 18 kDa expressed protein of porcine IL-18 was detected by SDS-PAGE; the specificity of expressed protein was confirmed by Western blotting. The purified porcine IL-18 protein induced obvious proliferation of porcine T lymphocytes in vitro, which indicated that the expression of IL-18 had high biological activity.

Gene cloning, expression and activity detection of porcine interleukin-18 mature protein in Escherichia coli / 生物工程学报

Porcine interleukin-18 mature protein gene was amplified from porcine spleen cells by RT-PCR. PCR product was cloned into the T vector pGEM-T for sequencing. The nucleotide sequence of this gene was 474 bp. Then, it was subcloned into the prokaryotic expressing plasmid vector pGEX6P-1 and transformed into host E. coli strain BL21 for expression. The expression of pIL-18 mature protein gene was identified by SDS-PAGE .The expression product was fusion protein with molecular weight of 45 kD and the percentage of expression protein in E. coil protein was 28%. The protein was purified by washing of inclusion bodies and the activity was measured by methyl thiazolyl tetrazolium (MTT).

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.

Construction of eukaryotic express vector of duck interleukin 18 gene and identification of bioactivity of its expressed protein / 生物工程学报

Duck IL-18 gene was amplified from plasmid pGEM-DuIL-18 by PCR. The PCR product digested with Pst I and Xho I was inserted into eukaryotic express vector pcDNA3.1(+) to generate an recombinant expression plasmid pcDNA3.1/DuIL-18 (pDuIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by restriction enzyme digestion, PCR and sequencing. DNA sequence confirmed the correct sequence of the recombinant eukaryotic expression plasmid pDuIL-18 in the reading frame and the ligation part. After the transfection of pDuIL-18 into Cos7 cells, duck IL-18 mRNA was expressed in Cos7 cell. The SDS-PAGE analysis showed that the expressed duck IL-18 protein had molecular weight of 23 000 D. The results of methyl thiazolyl tetrazolium (MTT) assay showed that duck IL-18 protein expressed in Cos7 cell could induce significantly transformation of duck T lymphocytes. Immunoenhancement effect of recombinant expression plasmid pDuIL18 on avian influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response.

Live-attenuated Salmonella typhimurium as a Oral Delivery Vehicle for SARS-CoV N DNA Vaccine / 中国生物工程杂志

Objective:To investigate the immunogenicity of SARS-CoV N DNA vaccine and the feasibility of live-attenuated Salmonella typhimurium as the carrier to deliver the N DNA vaccine.Method:The recombinant attenuated salmonella strain CS022 harboring the pcDNA-N DNA vaccine was constructed.And mouse was immunized with the recombinant strain via intranasal and oral routes.Cellular and humoral immune responses were assessed by ELISA,lymphocyte proliferation assays,ELISPOT and FACS.Result:The oral immunization with the transformed salmonellae elicited strong immune responses mainly including high level of N-specific antibody,a dramatic activation of IFN-?-secreting cells,a high level of lymphocyte proliferation,and a high level of activated CD8+ T cells.Conclusion:Live-attenuated Salmonella typhimurium could effectively deliver the SARS-CoV N DNA vaccine in vivo.These encouraging pre-clinical data provide a rational basis for undertaking a new immune style to investigate SARS vaccine.