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Abstract

Phosphorothioate CpG oligodeoxynucleotides (ODN) are reported to be recognized by the membrane-bound TLR9 and trigger the MyD88-dependent up-regulation of Type I interferons and pro-inflammatory cytokines. Whether plasmids containing multiple CpG motifs stimulate the same signaling pathway is yet to be determined. The present results show that the CpG motifs enrich plasmid pUC18-CpG stimulates RAW 264.7 in vitro, mainly through the TBK1-mediated signaling pathway, causing the up-regulation of IFN-β, and pro-inflammatory cytokines TNF-α and IL-6. When pUC18-CpG is co-administered with the recombinant Echinococcus granulosus antigen, the antigen-specific antibody titers are markedly increased compared to the Quil-A adju- vanted group. Antigen specific cytokine quantification shows that cytokine profiles from the pUC18-CpG adjuvanted-group are switched to a Th1-biased immune response.

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Authors and Affiliations

J. Wu
Q. Chen
T. Xin
Y. Sun
H. Jia
S.H. Hou
X.Y. Guo
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Abstract

An eukaryotic expression system of Congjiang pigs IFN-λ1 was constructed to obtain its expression in CHO-K1 cells and the inhibition effect of Congjiang pig IFN-λ1 on PRRSV proliferation was verified. The eukaryotic expression plasmid pEGFP-PoIFN-λ1 was constructed from the pig IFN-λ1 gene fragment and transfected into CHO-K1 cells. Expression was detected by fluorescence microscopy and Western blotting. The influence on the proliferation of PRRSV was assessed. The results of the study showed that the recombinant plasmid pEGFP-PoIFN-λ1 was constructed correctly. After transfection, green fluorescent signal was detected in CHO-K1 cells by fluorescence microscopy. Western blot analysis revealed that in cells at different time periods after transfection, porcine IFN-λ1 was expressed, with the highest expression observed 36 h after transfection. The antiviral activity of the supernatant after 36 h of transfection was determined by the micro cytopathic inhibition method, and the biological activity was 2.1×103 U/mL. Quantitative PCR was used to detect the proliferation of PRRSV, and the results showed that Congjiang pigs IFN-λ1 significantly inhibited the mRNA expression of PRRSV and viral proliferation in a dose- and time-dependent manner. This study established a Congjiang pig IFN-λ1 eukaryotic expression system, and the quantitative PCR method showed that it has a significant inhibitory effect on the proliferation of PRRSV, which lays a foundation for the future production of antiviral drugs and clinical application.

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Authors and Affiliations

J. Xin-qin
W. Biao
D. Zhi-qiang
R. Yong
H. Yan
H. Meng-qiu
D. Shan-shan
T. Yu-jie

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