Clone Of Porcine Nf-κB C-Rel Genes And C-Rel/P50/P65 Impacts On PRV Proliferation

The nuclear transcription factor-κappaB （NF-κB） is a general transcription factor in eukaryotic cell, the signaling pathway of it plays an important role in the bacterial and viral infection. NF-κB regulates innate immune response of host and roliferation, differentiation apoptosis of cells directly,and it’s abnormal activation maybe the key role to lead the immune suppression of PRV infection. In order to obtain sequence characteristics of Porcine NF-κB C-Rel gene and explore the relation of NF-κB abnormalities expression with immune suppression be caused of PRV infection. Clone, Sequence Identification, eukaryotic expression of NF-κB C-Rel（accession no. XM₀03125115） which was registered to NCBI Gene Bank.This study also discussed C-Rel gene transcription profiles in BHK21 infected with PRV in vitro, and its eukaryotic expression products on PRV proliferation. The results follow as:1. Cloning, Sequence Identification of Porcine Nuclear Transcription Factor-KappaB C-Rel genes NF-κB C-Rel ORF was amplified from pig peripheral blood lymphocytes and cloned,the sequencing results were analysed by some bioinformatics softwares. Sequence analysis showed that the C-Rel ORF contained 1 773 nucleotides in full length encoding a protein of 590 amino acid residues. The nucleotide sequence of porcine C-Rel genes shared a high similarity with other animals. C-Rel mainly located in the nucleus （76.0%）,no signal peptide and transmembrane region.2. Transcription Profiles of C-Rel in PK-15 Infected with PRV in Vitro This study use the web Primer3 Input, to design a pair of fluorescence quantitative primers with considering the C-Rel gene from NCBI,and built C-Rel gene fluorescence quantitative detection method successfully.The mRNA levels of C-Rel genes was analyzed using real-time fluorescence quantitative PCR（real-time FQ-PCR）. The real-time FQ-PCR results indicated that, the transcription levels of C-Rel mRNA were downing at 0-4 h after infected PRV, with significant differences at 2 h and 4 h （P<0.05）; then showed a different increasing levels at 4-12 h,and the transcription reached the highest level at 12 h （P<0.01）; subsequent transcriptions were downregulation rapidly, and had been at a low level in the 24-120 h after infected, with significant differences at 36 h （P<0.01）,48 h （P<0.05）,72 h （P<0.05）and 120 h （P<0.05）. It is thus clear that the infection of PRV could pull down the activation of NF-κB in PK-15.3. Construction of Eukaryotic Expression Vector of Porcine Nuclear Transcription Factor-kappa B C-Rel Submits and C-Rel/P50/P65 Impacts on PRV Proliferation The NF-κB C-Rel ORF was cloned to the eukaryotic expression vector pCDNA3.1（+）.Through the western-blot, C-Rel/P50/P65 were confirmed to express in cell nuclear after transfection pCDNA3.1（+）-C-Rel、 pCDNA3.1（+）-P65、pCDNA3.1（+）-P50-RHD.A strain of PRV named SCS was used to infect BHK21 cells that were transferred, and the supernatant was collected at different times postinfection to determine the influence of PRV multiplication from a set of three NF-Kb subunits including C-Rel,P50,P65. Fluorescent quantitative PCR was used to analyze PRV RNA quantity in different samples, and the One-step growth curve of SCS was determined according to the results.The eukaryotic expression vector can be expressed in BHK21 cells;the CPE was happened in advance because of transfection C-Rel/C-Rel、C-Rel/P50-RHD、C-Re/P65 eukaryotic expression plasmid;the multiplication of PRV in BHK21 was controlled because of transfection C-Rel/C-Rel、C-Re/P65 eukaryotic expression.