Prokaryotic Expression Of Marek’s Disease Virus Gene UL14and Dynamic Regulation In Vivo And In Vitro

Marek’s disease (MD) is a highly contagious and infectious diseases which is caused by MDV. It can cause internal organs and skin tumors and damage the central nervous system. MD is an ideal model for the study of neoplastic disease. UL14protein is a tegument protein that encoded by the Marek’s Disease Virus (MDV), which is highly conserved in the herpesvirus family. Some of the encoding region of the gene UL14overlaps that of UL13, which encods variable-length C-terminal domain. The most conserved residues are located in the nonoverlapping region. For herpes simplex virus type1(HSV-1), UL14protein not only can block apoptosis, but also has heat shock protein (HSP)-like functions and regulates viral replication. In this study, we detected the dynamics of UL14gene expression during MDV infection by Gene Chip and Real-time PCR. The results showed that UL14expression level was significantly different. We also expressed the UL14fusion protein in E. coli and made anti-serum to UL14.Part1:Marek’s disease virus gene transcription and expression patterns in chicken thymusIn this study, Gene Chip and Bioinformatics analysis were used to detect changes of virus gene levels of RB1B at7days post infection (dpi)>14dpi、21dpi、28dpi. Atotal of86viral genes were detected. At7dpi, there were47viral genes which could encod tegument protein, glycoprotein and nucleocapsid. Most of the genes were significantly over-expressed at21or28dpi and low expression level at14dpi. These results suggested that latent infection in the thymus at14dpi. In this phase, there were37viral genes were detected.8viral genes encoded tegument proteins (UL11, UL14, UL21, UL36, UL37, UL46, UL47and UL49) were detected. The UL14expression was significantly upregulated at21dpi (P<0.01) and downregulated at14and28dpi. This was consistent with the expression of Meq. The analysis of evolutionary tree showed that structure of UL14was similar to HSP70and had a high homology in the herpesvirus family. The current study provides transcriptomics information that will support further explain for the mechanism of viral pathogenesis and carcinogenesis.Part2:Prokaryotic expression of UL14gene and preparation of anti-serum against UL14protein To investigate the biological characterization of UL14protein during the course of MDV infection, DNA fragment of UL14gene from CEF cells infected with RBIB strain was amplified by PCR. The UL14cDNA was cloned into pET-32a (+) vector and expressed in E.coli BL21(DE3). The results showed that the recombinant His-UL14protein was expressed in E.coli and was soluble. Only a band of molecular weight of55kDa was purified by ion-exchange chromatography. Western blot indicated that the anti-serum to UL14could react with UL14fusion protein. IFA result confirmed that the anti-serum from BALB/c mice immunized with the recombinant protein could react with CEF infected with RB1B strain.Part3:Dynamics analysis of UL14gene expression of MDV in vitro and in vivoTo investigate the dynamics of UL14gene expression, we detected the UL14gene exprssion level in chicken tissues and CEF cells infected with vaccine strain CVI988or very virulent strain RBIB by Real-time PCR. The results showed that UL14gene expression was increased from24hours post infection (hpi) to72hpi then decreased in virus-infected cells. This result was consistent with western blot. Results of animal experiment showed that UL14had same expression pattern in RB1B-infected chicken spleen and thymus. The UL14expression was significantly upregulated at21dpi (P<0.01) and downregulated at14and28dpi. The expression showed a wavelike pattern, which was down-up-down during MDV RBIB infection. This was consistent with expression of Meq. Unlike RBIB infected spleen and thymus, we found that the highest level of UL14exprssion in CVI988-infected chicken spleen and thymus was at14dpi (p<0.01) and then decresed gradully. Our results gave a sund data to study the UL14pathogenesis of Marek’s disease virus.