miR-365, a novel negative regulator of interleukin-6 gene expression, is cooperatively regulated by Sp1 and NF-kappaB.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in host defense. IL-6 expression can be regulated at both a transcriptional and a post-transcriptional level. We used a combination of bioinformatics and experimental techniques to demonstrate that the miR-365 is a direct negative regulator of IL-6. Overexpression of miR-365 mimics decreased activity of a luciferase reporter containing the IL-6 3'-UTR and led to repression of IL-6 protein. In contrast, ectopic expression of a miR-365 inhibitor elevated IL-6 expression. The negative regulation of miR-365 was strictly dependent on a microRNA binding element in the 3'-UTR of IL-6 mRNA. Deletion mutant analysis of the miR-365 promoter showed that two transcription factors, Sp1 and NF-κB, are essential for the transcriptional regulation of miR-365. We also demonstrate that the MAPK/ERK pathway contributes to the regulation of miR-365. Furthermore, miR-365 exhibited a greater negative regulatory effect on IL-6 than hsa-let-7a, a previously identified microRNA negatively regulating IL-6. Taken together, our results show that miR-365 is a novel negative regulator of IL-6.

[Co-expressed GP5 and M proteins of porcine reproductive and respiratory syndrome virus can form heterodimers].

In order to investigate the characterization of in vitro co-expressed GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV), eukaryotic expression plasmids pCI-ORF5 (expressing GP5 protein alone), pCI-ORF6 (expressing M protein alone), and pCI-ORF5/ORF6 (co-expressing GP5 and M proteins) were constructed. After transient transfection, Western blot analysis under nonreducing condition demonstrated that co-expressed GP5 and M proteins could form disulfide-linked heterodimers (GP5-M) in transiently transfected BHK-21 cells. To further study the influence of GP5-M heterodimers formation on the subcellular localizations of GP5 or M proteins, green fluorescence protein (EGFP) and red fluorescence protein (RFP) were used as markers. The results of fluorescence distribution showed that co-expressed GP5-EGFP chimera and M-RFP chimera boosted the transport of GP5 from the endoplasmic reticulum (ER) to the Golgi complex, indicating that the formation of GP5-M heterodimers may be involved in posttranslational modification, transport, and subcellular localization of GP5. These results presented here lay foundation to further study the molecular mechanism of GP5-M heterodimer formation and its role in protective immunity of PRRSV.

[Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1].

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.

[Bias of base composition and codon usage in pseudorabies virus genes].

The complete sequence of the Pseudorabies Virus (PRV) genomic DNA has not yet been determined, primarily because of the high content of G + C nucleotides of about 74%. We examined the base composition and codon usage of the 68 known PRV genes. As a result, we found a strong bias towards GC-rich codons especially NNC or NNG (N represents any one of four nucleotides) in PRV genes. This demonstrated that the usage bias of synonymous codon and amino acid is the main cause of the high G + C content of PRV. The results showed that the genome regions adjacent UL48, UL40, UL14, IE180 genes where the G + C content occurs as pronounced waves are corresponding to the replication origins. It was also found that the codon usage patterns of regulatory genes are apparently different from other PRV genes. A corresponding analysis of amino acid compositions indicated that the bias of codon usage could be related to the differences of gene function.

[Expression of GP5-M fusion protein of porcine reproductive and respiratory syndrone virus (PRRSV) and establishment of ELISA diagnose based on the recombinant fusion protein].

The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.

[High expression of the foot-and-mouth disease's structural protein P1 in Escherichia coli and analysis of its biology activity].

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.

[Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus].

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.

[Construction and growth properties of recombinant pseudorabies virus expressing modified enhanced green fluorescent protein].

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence w as amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of P RV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/LacZ(+), was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p.i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24-36 h p.i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG(-)/M1(+) in tissue cultures were further examined and the titer of gG(-)/M1(+) was similar to that of PRV Ea wild strain and the parental strain gG(-)/LacZ(+). The above results indicated that the recombinant virus expressing the modified EGFP can be used as an in vivo marker to monitor replication and spread, as well as t o study the molecular pathogenesis of PRV.