Dynamic Changes Of Expression Levels Of BLec、BNK And Cytokines In PBLs Of B19and B21Haplotype SPF Chickens Infected With MDV

Major histocompatablity complex(MHC) has long been thought to be associated with immune response system of recognition susceptible and tolerance to Marek’s disease (MD). Six haplotypes, including B13, B15, B2, B5, B21and B19chickens have been screen bred based on four microsatellite loci of MHC-B region in National Laboratory Animal Center of Harbin Veterinary Research Institute. In this experiment, we used B15,B19and B21haplotype chicken.In this study, total RNA was extracted from one two months-old B15haplotype chicken thymus tissue and ChBLee gene was amplified by RT-PCR following by cloning into pET-30a (+) vector, prokaryotic expression recombinated plasmid of pET-30a-ChBLec was successfully. After being transformed into E. coli BL21(DE3) the ChBLec recombinant protein was induced to express using IPTG (0.02mmol/L). Rabbit antiserum was obtained.The result of western blottiing showed that recombinant protein about17kDa.To study dynamic changes of BNK, BLec and four cytokines in B19and B21haplotypes chicken PBL. One day-old chickens were infected with MDV Md5strain by intramuscular as800PFU per feather, the control group using steriled PBS. PBL was collected on4,7,10,13,19dpi and separated, then RNA was extracted after stimulation with ConA (25ug/mL) for48h. The content of BNK, BLec and four cytokines were detected by RT-QPCR. The result showed that:on4,7and19dpi.transcript levels of BNK and BLec was decreased; On10dpi transcript levels of BNK and BLec in B19haplotype chicken was increased, transcript levels of BNK in B21haplotype chicken was increased; on13dpi transcript levels of BNK said BLec in B21haplotypes chickens was increased. Dynamic changes of four cytokines in different time points showed that transcription level of four cytokines on4,7and19dpi was decreased; on10dpi, transcript levels of IFN-y (19%), IL-4(5%), and IL-18(32%) in B21haplotype was increased, above three cytokines (-16%,-29%,-23%) of B19haplotype was decreased. On13dpi,transcript levels of IL-18(47%) and IL-10(69%) in B21haplotype chicken was increased, transcription levels of IL-18(-30%) and IL-10 (-37%) in B19haplotype chicken was decreased.According to the literature, the pathogenesis of MD shows that lymphoid organs will be atrophy in the first cytolytic infection (4,7dpi), resulting in immunosuppression. In the experiment, transcript levels of BNK, BLec and cytokines on4,7dpi was decreased; chicken lymphoid organs was recovery after8-14d infection, and generally tolerated chickens is not subjected to a second cytolytic infection.On the contrary susceptible chickens will continue immunosuppression in the second cytolytic phase. On10,13dpi transcription levels of BNK, BLec and cytokines in tolerated chicken was increased,because lymphoid organs was recovery. These results showed that this change is associated with chicken genetic background. Chicken B21haplotype is tolerated than chicken B19haplotype.