Expression Of UL13 Gene In CVI988 Rispens Strain Of Marek's Disease Virus And Preparation Of The Monoclonal Antibodies Against UL13

The UL13 gene of Marek's disease virus encodes a tegument of approximate 57kDa~60kDa, a homology with the herpes simplex virus type 1(HSV-1) UL13 gene and varicella-zoster virus ORF47. It's has been verified that UL13 protein (pUL13) of HSV-1 and ORF47 protein encode a viral protein kinase, which could phosphorylate several viral proteins and cellular protein in vitro or in vivo.To investigate the function of MDV UL13, a panel of monoclonal antibodies specific to UL13 protein of MDV was developed by fusion between SP2/0 cells and spleen cells from the mice immunized with purified UL13 protein by electro-elution, which was a truncated protein expressed in Ecoli. These monoclonal antibodies could react with recombinant UL13 protein expressed in Sf9 cells. These materials could be very good tools for further investigating MDV UL13 gene functions.1 cloning and sequence analysis of MDV UL13 geneAccording to UL13 gene sequence of MDV GA strain in Genbank, a pair of specific primers was designed and synthesized. UL13 gene was amplified from the whole DNA of Chicken embryo fibroblasts cells (CEFs) infected with MDV CVI988 strain. The PCR product was inserted into the pGEM T easy vector and named as pGEM-UL13. Sequencing analysis showed that the UL13 complete open reading frame was obtained, which is 1542bp, encodes 513 amino acids. Comparing with other members of herpesviridae, the results showed 99.8% of homology in deduced amino acids between Gallid herpesvirus 2. Only one point mutant was found at 192 site:(Arg→Gln). However, MDV UL13 gene showed merely 9.1%~66.8% of homology with some other members of herpesviridae, but they shared several protein kinase conserved domains. Analyzing the MDV UL13 protein in the EXPASY database, several protein kinase conserved domains have been found such as Gly residue at 152,154,157 site, Val residue at 159 site and Lys residue at 170 site within the polypeptides 151AGSGSYGVV159 and 168AVKK171, which are responsible for functions of ATP binding. Peptide 264LTHLDIKCGNIFV276 maybe has features of Serine/Threonine protein kinase active site. Asp residue at 268 site is as the proton acceptor in this motif. These results indicated that the MDV UL13 gene is a putative protein kinase and has Serine/Threonine kinase activities.2 Prokaryotic expression and preparation of polyclonal antibodies against MDV UL13UL13 gene of CVI988 and the truncated fragments were inserted into prokaryotic expression vector pGEX-6P-1, fusion with a gene encoding glutathione S-transferase (GST), respectively named as pGEX-UL13, pGEX-UL13N (1-114aa), pGEX-UL13C (115-513aa), pGEX-UL13S (260-400aa) and pGEX-UL13H (432-513aa). These plasmids were transformed into the competent cells of BL21 (DE3) E.coli. After 2.5h incubation, expression of UL13 and truncated UL13 protein were induced by 0.05mmoL IPTG. After brief sonication and centrifugation, the supernatant and precipitation of the cell lysate were separated by 12% SDS-PAGE. Then expressed fusion proteins were identified by Western blot. Several fusion proteins, GST-UL13C, GST-UL13S and GST-UL13H, were expressed successfully. The fusion protein was excised and recovered by electro-elution. The mouse was immunized with four injections at 2-weeks intervals, each injection containing 100μg of purified antigen. Seven days after the final immunization, the serum was collected. The results of western blot assay for the truncated UL13 protein and GST expressed in E.coli showed that the serum could react with fusion proteins. This means electro-eluted truncated UL13 protein can effectively stimulate immune response in mice.3 Expression and Identification of MDV UL13 in insect cellsUL13 gene was digested from the pGEM-UL13 vector and inserted into pFastBac1TM vector. After identification by EcoR I and BamH I enzyme digestion, the recombinant was transformed into the competent cells of DH10Bac Ecoli which contain the bacmid with a mini-attTn7 target site and the helper plasmid. The recombinant bacmid-UL13 was generated by transposing the mini-Tn7 element located in pFastBac1-UL13 to the mini-attTn7 attachment site on the bacmid. Then the recombinant Bacmid DNA was extracted and transfected into the Sf9 insect cells mediated by lipofectin2000TM to generate recombinant baculovirus. The UL13 gene in recombinant baculovirus was amplified by PCR from the extract of culture supernant and the UL13 protein expression in insect cells was verified by IFA and Western blotting at 4 days post-transfection. These results indicated the UL13 protein expressed in insect cells.4 Preparations of the Monoclonal Antibodies against UL13The spleen cells of Balb/c mice immunized five times with electro-eluted GST-UL13C protein expressed in Ecloi were fused with SP2/0 myeloma cells. After 10 days, the cell culture medium of hybridoma cells was detected with sf9 infected with rBacmid-UL13 baculovirus by IFA. Four hybridoma cell lines secreting monoclonal antibodies (McAbs) against MDV UL13 were developed with limited dilution method, respectively named as MDV-UL13-4H9,MDV-UL13-4G11,MDV-UL13-5D12,MDV-UL13-2A4. IFA analysis showed that these McAbs could specifically react with the sf9 insect cell infected with rBacmid-UL13, but not with sf9 infected with rBacmid-NP (AIV) or rBacmid-VP2 (IBDV). The preparation of monoclonal antibodies against MDV UL13 will be useful materials for studying the biological functions of the protein.