Expression Of Porcine Nuclear Transcription Factor-κappaB P65/p50Genes And Its Transcription Profiles In Marc-145Infected With PRRSV And Its Impacts On PRRSV Proliferation

The signaling pathway of Nuclear transcription factor-κappaB （NF-κB） plays an important role in the viral infection and the process of disease development, it can regulates host innate immune response and cell proliferation, differentiation and apoptosis, and NF-κB abnormal activation is maybe the key role to lead the immune suppression of PRRSV infection. In order to obtain sequence characteristics of Porcine NF-κB p65, p50submit gnes and explore the relation of NF-κB abnormalities expression with immune suppression be caused of PRRSV infection. Clong, Sequence Identification, prokaryotic/eukaryotic expression, protein purification and preparing polyclonal antibodies of NF-κB p65（accession no. NM₀01114281）, p50（accession no. NM₀01048232） which were registered to NCBI Gene Bank, and its transcription profiles in Marc-145infected with PRRSV in vitro, and its prokaryotic/eukaryotic expression products on PRRSV proliferation were studied. The results follow as:1. Clong, Sequence Identification of Porcine Nuclear Transcription Factor-KappaB p65/p50genes NF-κB p65ORF, mature p50coding region,and p50-RHD were amplified from pig peripheral blood lymphocytes, then cloned into the pMD19T simple vector and sequences characteristics were analysed. Sequence analysis showed that the p65ORF contained1662nucleotides in full length encoding a protein of533amino acid residues, the p50mature protein coding region contained1653nucleotides encoding551aa, the p50-RHD coding region contained777nucleotides encoding259aa. The nucleotide sequence of porcine p65/p50genes shared a high similarity with other animals. p65majority located in the nucleus （56.5%）, p50majority existed in the cytoplasm （78.3%）, no signal peptide and transmembrane region neither.2. Prokaryotic expression of pig nuclear transcription factor-kappaB p65/p50Preparation of Anti-p65/p50Antibody The p65ORF, encoding mature p50gene and p50-RHD gene were connected into the pET-21a（+）/pET-32a（+） vector, then transformed into Escherichia coli Rosetta （DE3）, recombinant protein were expressed by IPTG, the expression products were identified by SDS-PAGE and Western-Blot. The polyclonal antibodies were prepared by by immunizing rabbits with purified renatured p50/p50-RHD/p65recombinant protein, and polyclonal antibodies titers were detected using the agar diffusion test. The results showed that p50/p50-RHD/p65recombinant protein were expressed abundantly in the form of inclusion bodies with molecular weight of70KD/45KD/70KD respectively, Western-Blot results showed that the rabbit anti-human p50polyclonal serum, rabbit anti-human p65purified antibody can bind particularly with p50and p65respectively, the antibody titers of rabbit anti-p65, rabbit anti-p50-RHD, rabbit anti-p50serum were1:8,1:16,1:32respectively.3. Influence analysis of NF-κB p65/p50Transcription Phase and Nuclear Translocation in Marc-145cells infected with PRRSV in vitro The p65/p50genes mRNA transcription levels of Marc-145cells infected with PRRSV in different periods were analyzed using real-time fluorescence quantitative PCR（real-time FQ-PCR）. Nuclear proteins of Marc-145cells infected with PRRSV in different periods were extracted, and the activity levels of p65/p50in cell nuclear were detected by Western blot. Real-time FQ-PCR results indicated that, the p65/p50mRNA transcription levels of Marc-145cells decreased rapidly8-12hours post infection（hpi）, then keeping at a low level12-120hpi, which was significantly lower than the levels of control group at the same time and Marc-145cells without PRRSV infection（P<0.05or P<0.01）. The p50expression in cell nuclear could not be detected12-96hpi by Western blot, neither nor the p6512hpi. The results revealed that PRRSV infection in vitro would inhibite NF-κB p65/p50mRNA transcription and nuclear translocation. The PRRSV-induced immunologic suppression may be related to the low activation level of NF-κB. This study would provide some new ideas for further study on the pathogenic mechanism of PRRSV and the function of NF-κB..4. The impacts of NF-κB p65/p50Prokaryotic expression products on PRRSV proliferation Added the purified and refolded p65/p50to the2%FBS DMEM（Cells maintain nutrient solution）, observed its cytotoxicity on Marc145to selecte the optimum concentration. The effects of optimum concentration p65/p50on PRRSV proliferation activity were studied by detecting PRRSV infection phase in the culture supernatant using real-time FQ-PCR method and drawing PRRSV one-step growth curve. The real-time FQ-PCR results indicated that, NF-κB p65/p50can promote CPE appearance and PRRSV proliferation before CPE appeared, then suppressed PRRSV proliferation after CPE appeared, and lower the virus titer levels significantly（P<0.05）.5. The PRRSV proliferation in Marc145transfected the eukaryotic expression vectors of Porcine NF-κB p65/p50subunits:The eukaryotic expression vector of NF-κB p50, p65subunits were constructed, then the two recombinant plasmids were transfected into Marc145to study the impacts on PRRSV proliferation Simultaneously or separately. Transfected p50+p65, p65eukaryotic expression plasmid would promote PRRSV infection Marc145CPE appears to accelerate the CPE process, eventually PRRSV proliferation was inhibited, the PRRSV replication inhibition capacity of p50+p65was stronger than p65. Transfected p50eukaryotic expression plasmid would postpone the PRRSV infection Marc145CPE appears to slow down the process of PRRSV proliferation in Marc145cell, but no significant effects on the level of virus titer.