Expression Of Viral Interleukin 8 Gene Of Marek's Disease Virus And Its Biological Characterizations

Marek's disease virus (MDV) is a herpesvirus which induces the tumor of T cells in chickens. However its oncogenic mechanism is still unclear. In this study, total RNAs were respectively extracted from chicken embryo fibroblast (CEF) cells infected with MDV reference strain 648A, RB1B, MD11, CVI988, Z4 and HVT, and the cDNA sequences of viral interleukin 8 (vIL-8) were amplified by RT-PCR. The results showed that only the cDNA about 430bps of serotype I of MDV could be amplified, but nothing could be found in serotype II > III of MDV. The PCR products were cloned into pGEM-T easy vector (pT-vIL8) and sequenced. We found that cDNAs of Strain- 648A, RB1B, MD11 and CVI988 were consistent with Strain-GA in sequence analysis with DNAstar software. These results suggested that vIL-8 gene sequence be high conserved, and could be good marker in identifying type I of MDV from others.To understand the biological activity of vIL-8, we took down vIL-8 gene with restriction endonucleases EcoR I and BamH I from pT-vIL8. The fragment was inserted into expression vector pGEX-5X-3 digested with the same enzymes. The recombinant expression vector was constructed (pGEX-vIL8). Then host cells of BL21 Escherichia coli (E.coli) were transformed with pGEX-vIL8. The result showed that the recombinant vIL-8-GST product was expressed in BL21 E. coli induced by IPTG. The fusion protein of vIL-8-GST showed attraction to chicken peripheral blood lymphocytes in Chemotaxis assay. It suggested that vIL-8 might attract susceptible cells to improve infection and take a role in tumor spreading and progressing during the course of MDV infection.The vDL-8 gene fragment was inserted into eukaryotic expression vector pcDNA3.l(+) /zeo, named pcvIL-8, which was transfected into host cell COS-1. The supernatantof tissue culture cell transfected with recombinant plasmid pcvIL-8 post 48 hoursshowed strong chemotaxis activity to peripheral blood mononuclear cells of chickens.The titration of the activity was 105 . These results demonstrated that the COS cell hadsecreted vIL-8.The cytokines often have the effect of immune-regulation, especially in gene immunization. To make sure whether vIL-8 has the same characterization, we mixed the supernatant of COS-1 cells transfected with pcvIL-8 with red blood cells type O of human (hRBC) .And then the mixture was injected into mice through i.p. The levels of antibodies to hRBC from the mice serum were detected. The result showed no difference between the experimental group and control group.In addition, the nake plasmid of pcvIL-8 and pcALV-Jenv were mixed and injected into chickens by i.m. The tilers of antibodies to ALV-J env from the chickens serum were detected and no difference were found between different groups. Chickens immunized with ND vaccine (Lasota strain) mixed with plasmid pcvIL-8 by injection muscle showed the same titration of antibodies to KDV in HI assay. These results revealed that vIL-8 might not play a role in regulation of humor immunity. However other effect of immune-regulation will be investigated in future.