| Classical swine fever (CSF) is a kind of highly contagious and lethal pig infectious disease, caused by classical swine fever virus (CSFV), which is characterized by haemorrhage and hyperthermia. Usually, the CSFV infected pigs will be massively died and leads to huge economic losses. CSFV is a single strand RNA virus, belongs to Flaviviridae family, Pestiviruses genus. The non-structural protein (Npro) is the first protein which is translated by CSFV genome. Npr0 possesses the auto-protease activity of CSFV and it can catalyze the polyprotein to become 12 mature viral proteins. Npro functions the inhibition of interferon regulatory factor 3 (IRF3) transcribed by IFN-a/p, leading to that IFN-α/β could not induce the downstream factors. Interferon regulatory factor 1 (IRF-1) is an important multifunctional transcription factor. The IRF1 family possesses a variety of functions such as modulating the expression of interferon (IFN) and has anti-viral or anti-tumor activity. In addition, IRF1 selectively modulates the immune responses of different genes in different kind of cells as well. In this study, on the basis of previous work, we would like to further exploration of the interaction between CSFV and host. However, there is not relative antibody (Ab) such as anti-Npro or anti-IRF-1 Ab as commercial product on sales on the biological market. Therefore, we develop anti-Npro and anti-IRF-1 antibodies to pursue further researches on the structure and functions of CSFV proteins. And the preparation of the antibodies is able to lay the foundation for analysis the interaction of IRF-1 with IFN, interferon stimulate gene (ISG), and other immune response factors.In this study we had successfully constructed the recombinant prokaryotic expression plasmid pET-Npro and pET-IRF-1 by cloning Npro and IRF-1 gene into PMD18 vector, and then sub-cloning into prokaryotic expression vector-pET-32α. The results showed that the pET-Npro and pET-IRF-1 were induced to express in E.coli Rosetta, and to prove that both Npro and IRF-1 gene had effectively expressed. The Npro is expressed either in a form of inclusion body or in a form of soluble protein. However, IRF-1 is mainly expressed in a form of soluble protein. Both recombinant Npro and IRF-1 proteins were purified by the nickel-ion (Ni+) affinity chromatography and used as antigen to prepare polyclonal antibody (PAb) by administrating 4 week-age mice according to the immune procedure. As a result, both anti-Npro and anti-IRF-1 PAbs reacted well with recombinant proteins Npro and IRF-1, respectively by Western blot. We also found that the anti-Npro PAb was able to bind to the native conformational Npro in the CSFV infected PK-15 cells. In addition, the IRF-1 was induced in the CSFV infected PK-15 cells at 48h. Both anti-Npro and anti-IRF-1 PAbs were capable to react to Npro and IRF-1 in eukaryotic PK-15 cells specifically. Taken together, both anti-Npro and anti-IRF-1 PAbs were identified to have highly specificity and reactivity to the Npro and IRF-1 proteins, respectively.We found that the mRNA level of IRF-1 in PK-15 cells was increased during CSFV infection by real time quantitative PCR. And the IRF-1 protein was obviously induced by CSFV by Western blot. The results proved that CSFV could increase levels of IRF-1 expression. These results suggested that IRF-1 might probably play an important role in the CSFV infection. |
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