Expression Of UL41 Gene Of Marek's Disease Virus CVI988/Rispens Strain And Preparation Of The Polyclonal Antibodies Against UL41

The UL41 gene of Marek's disease virus (MDV) with high homology to that of the herpes simplex virus type 1(HSV-1) encodes a stromatin with approximately 50kDa. Previous data shown UL41 protein of HSV-1 not only has the nuclease activity, but also was involving in the pathogenisis and the immune escape. To understand whether the UL41 gene from the MDV attenuated vaccine strain CVI988 play a role in the immune escape, UL41 gene of CVI988 strain was first cloned and expressed, and the polyclonal antibody specific to the UL41 protein were developed in mice.1. Clone and analysis of UL41 gene from MDV CVI988Using specific primers, UL41 gene was amplified by PCR from the whole genomic DNA of the chicken embryo fibroblasts cells (CEFs) infected with MDV CVI988 strain. Senquenc analysis showed that the nuclear acid senquence of the gene had more than 99.8% identity and amino acids sequence induced had more than 99.3% identity among MDV-1 strains. Moreover, UL41 protein shares the significant XPGI domain with a number of eukaryotic cellular nuclease.2. Prokaryotic expression of UL41 protein of MDV CVI988 and its polyclonal antibodyThe UL41 gene of MDV CVI988 was cloned into prokaryotic expression vector pGEX-6P-1, fusion with a gene encoding glutathione (GST). After incunation by IPTG, the supernatant of the pGEX-UL41 bacteria lysate was collected for SDS-PAGE and Western blot. The result showed that the UL41 was expressed successfully and the interest protein formed inclusion bodies. The fusion proteins were purified by electro-elution and were injected into mouse to produce polyclonal antibodies.3. Expression of UL41 protein of MDV CVI988 in recombinant baculovirus The UL41 gene was cloned into baclovirus vector pFastBac1 to construct pFastBac-UL41. Then pFastBac-UL41 was transformed into DH10Bac E.coli cells, and to identify the positive recombinant Bacmid DNA by PCR. The recombinant Bacmid DNA was transfected into Sf9 cells to generate recombinant baculovirus rBac-UL41. The expression of UL41 in rBac-UL41 was verified by IFA by using polyclonal antibody specific to UL41 protein.