Construction Of Recombinant Baculovirus And Cell Location Of Duck Enteritis Virus GH And UL24

Duck viral enteritis is an acute, feverous and blood poisonous disease of waterfowl (duck,goose and swan) caused by Duck Enteritis Virus. The 41 complete DEV ORFs were cloned at leastat present and the properties of gH and UL24 were studied in this paper.Construction of recombinant baculovirus of DEV gH and UL24: according to gH gene andUL24 gene datas of DEV published in Genbank, 3 pairs of primers p1/p2, p3/p4 and p5/p6 foramplification of gH gene and UL24 gene were designed, gH gene was divided into two overlappedfragments gH1 and gH2. gH1 gene was amplified with primers p1/p2, the PCR product was ligasedto pMD18-T vector to construct pMD18-T-gH1, gH2 gene was amplified with primers p3/p4.pMD18-T-gH1 was digested with Xhoâ… and Sfcâ… , and gH2 gene was digested by Sfcâ… and Kpnâ… .Subsequently the two overlapped fragments were subcloned to the same transfer vectorpBlueBacHis2A to constuct recombinant baculovirus transfer vector pBlue-gH. UL24 gene wasamplified with primers p5/p6 and the PCR product was ligased to pMD18-T vector to constructpMD18-T-UL24. The UL24 gene was then subcloned to the transfer vector pBlueBacHis2A toconstuct recombinant baculovirus transfer vector pBlue-UL24. Purified recombinant eukaryotictransfer vector pBlue-gH and pBlue-UL24 were transfected into sf9 cell with linear baculovirusseperately.The pure recombinant baculovirus pBlue-gH and pBlue-UL24 were acquired by threeround plague assayed.Cell location of gH protein: The PCR product of gH gene was inserted into pMD18-T toconstruct pMD18-T-gH and was digested with Hind and EcoRâ… as eukaryotic expression vectorpcDNA3.1 (+) did. The purified pcDNA3.1-gH was transfected to 293 cells with lipofectamine^TM2000 and BALB/c mouse were immuned with purified pcDNA3.1-gH to raise Anti-gH serum.Transient expression of DEV gH protein in vitro was detected by indirect immunofluorescenceassay and the results indicated that DEV gH protein localized in cytomembrane. The RNA wasextracted from transfected cells and identyed by PCR with specific primers of gH, the resultsfurther proved expression of gH protein.Cell location of UL24 protein: UL24 gene was amplified with primers p7/p8 designed basedon published DEV UL24 gene dates in Genbank. The PCR product was subcloned to pEGFP-C1 toconstruct recombinant expression vector pEGFP-UL24, which was used to transfect to 293 cellswith lipofectamine^TM 2000 then. The intracellular localization of UL24 protein was observed and the results indicated that UL24 protein localized mainly in nucleus. The RNA was extracted fromtransfected cells and identyed by PCR with specific primers of UL24, the results showed that UL24gene expressed transiently in vitro.