Identification Of Novel ORFs On Marek’s Disease Virus

Marek’s disease (MD) is a highly contagious disease, which is caused by Marek’s disease virus (MDV) and characterized as the infiltration of monocytes and the evacation of lymphomas. Meanwhile, MDV also cause immunosuppression, and then produce secondary infection. Poultry industry suffer enormous economic loss because of MD every year. With the completion of sequencing the genomewide of different MDV-1strains, people recognize the bionomics of MDV-1more clearly.The prediction of novel ORFs based on the condon usage of well-characterized mardivirus genes widen the way of researching the characterization of the biology of MDV-1.This paper identify mainly the ORFs that is specific to MDV-1based on the genome of CVI988/Rispens, which pave a way to research the mechanism of pathopoiesis, tumorigenesis, attenuation of virulence and immunosuppression.The ORFs that can code100aa and are specific to MDV-1were seleted, and ORF1.0/ORF80.0, ORF5.6/ORF75.9, ORF89.5, ORF91.5and ORF97.3were suitable. The antigenicity of selected ORFs were analyzed through the usage of DNAstar softeware and the segment of ORFs were amplified. The ORFs that could express in CEF were detected firstly by the method of RT-PCR, and the result showed only ORF5.6/ORF75.9, ORF91.5and ORF97.3could expressed in CEF which inocuted with CVI988/Rispens.The method of RT-PCR was used to amplify successfully the expected length fragment of ORF5.6/ORF75.9, ORF91.5and ORF97.3from chicken embryo fibroblast (CEF) inoculated with CVI988/Rispens, and then cloned into the expression vector pET-32a(+), respectively. After sequencing, the recombinant plasmid was transformed into BL21. The transformed bacteria were induced by IPTG and produced recombinant proteins of33kDa、36kDa and31kDa in mass, respectively. The rabbit antiserum against recombinant protein was produced by using recombinant proteins as the immunogen to immunize rabbit, and then by using the rabbit antiserum against recombinant protein as primary antibody, the method of IFA was used to confirm the ORF5.6/ORF75.9, ORF91.5and ORF97.3genes could be expressed in CEF inoculated with CVI988/Rispens. Howerver, the result of Western-blot showed that only the recombinant protein of ORF91.5could react the chicken antiserum against strain CVI988/Rispens, the ORF5.6/ORF75.9and ORF97.3could not react the chicken antiserum against strain CVI988/Rispens.To further identify whether ORF5.6/ORF75.9and ORF97.3expressed in chickens inoculate with CVI988/Rispens, the liver and spleen were collected at7d,14d and21d, and through RT-PCR method, we detected the mRNA of ORF5.6/ORF75.9and ORF97.3in chickens. The result demonstrated that ORF5.6/ORF75.9could expressed in14d and21d of spleen, but the quantity of mRNA of ORF5.6/ORF75.9were apparently decreased. Howerver, the mRNA of ORF5.6/ORF75.9only were detected in14d of liver. Moreover, the mRNA of ORF97.3was not detected in liver and spleen.The bioinformatics of ORF5.6/ORF75.9、ORF91.5and ORF97.3was analyzed, which only exised in MDV-1and there were not any homologous genes in HVT (the turkey of herpisvirus), MDV-2and HSV (herpesvirus simple virus). The deduced amino acid sequence of ORF5.6and ORF91.5gene were searched for protein motifs with PROSITE tools and the fuction of these two protein were searched too, but no any appropriate databases and motifs of these proteins were hit. Through the protein blast in NCBI, the homology of ORF5.6/ORF75.9and ORF91.5in different MDV-1strains was high, but the the homology of ORF97.3in different MDV-1strains contain regular mutations.ORF5.6/ORF75.9, ORF91.5and ORF97.3genes were first identified protein-coding genes by RT-PCR and immunofluorescence assay. Meanwhile, the basically characteristics of novel ORFs were researched by Western-blot, RT-PCR and the analysis of bioinformatics, which supplied some valuable information to research the biological feature of MDV-1.