Construction Of DEV Gene Library And Discover And Cloning And Expression Of Its Nucleocapsid Protein Gene

Duck viral enteritis (DVE), also named as duck plague (DP), is an acute, contagious herpesvirus infection of the anseriformes such as duck, geese and swans, characterized by vascular damage, tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs. In duck-producing areas of the world where the disease has been reported, DVE has produced significant economic losses in domestic ducks and wild waterfowls due to mortality, condemnations, and decreased egg production. The causative agent of DVE is duck enteritis virus (DEV), also called as duck plague virus (DPV) or Anatid herpesvirus 1 (AHV-1), which is a herpesvirus, belonging to the Alpha herpesvirus subfamily. In this study, DEV CHv strain, isolated from Sichuan province in 1999, was selected to study its purification and restriction endonuclease analysis of genome DNA. The gene library of DEV was constructed by shotgun way and the nucleocapsid protein gene was discovered in the library and identified by PCR and sequencing. A new method of PCR to DEV was developed on detecting its nucleocapsid protein gene, and this nucleocapsid protein gene was cloned into prokaryotic expression vector pET32a and expressed in E.coli BL21 (DE3). All studied were listed below:1. Purification of DEV CHv Strain and SDS-PAGE Analysis of Its Structural Proteins: After propagated in duck embryo (DE) and duck embryo fibroblast monolayer cells (DEF), DEV CHv strain was separately purified by four methods such assedimentation, chromatography, differential centrifugation and sucrose gradient centrifugation. The results indicated that the effect of purification of sucrose gradient centrifugation from viral culture medium propagated in DEF was the best, and purified virions with characteristic of herpesvirus were observed under transmission electron microscope by using negative staining of 2% phosphotungstic acid. And the banded virus was mainly located between 40%~50% sucrose gradient and the diameter of enveloped virions was 170nm~190nm. And eleven of viral structure proteins, VPl(190kD), VP2(136kD), VP3(106kD), VP4(88kD), VP5(75kD), VP6(68kD), VP7(56kD), VP8(48kD), VP9(42kD), VP10(38kD) and VPll(32kD) were revealed by SDS-PAGE and rapid Coomassie blue staining, and VP1, VP2, VP3, VP6, VP8 and VP9 were main structural proteins and accounted for 89.04% in total structural proteins.2. Restriction Endonuclease Analysis of DEV Genome DNA: The genome DNA of DEV propagated in duck embryo fibroblast monolayer cells (DEF) was extracted by two methods and digested by restriction endonucleases BamH I , Bgl II , EcoRM, H//7dII, Kpn I , Pst I , Sal I > Iba I and Xfio I respectively, and its DNA fragments digested were compared with those of other DEV strains. The results showed that the extracted effect of the DNA was better from the purified virions by sucrose cushion ultracentrifugation than from DEV infected DEF cells directly. Digesting with 9 restriction endonucleases, there were separately 12, 15, 12, 9> 14> 15, 12, 15 and 13 bands of DNA fragments by 0.8% agarose gel electrophoresis. The DNA fragments of DEV CHv strain genome had some differences in amount, molecular weight and total with that of other DEV strains. These results will facilitate the molecular virological study of DEV.3. Construction of DEV Gene Library and Discover and Identification of Its Nucleocapsid Protein Gene: Fragmentated by supersonic wave and separated by SMART cDNA Library Construction Kit of DEV genome DNA extracted from purified virion, the DNA fragments were ligased to pBluescript II SK* and transferred into competent cells of E.coli. DH5a. After the positive recombinant colonies were selected by the color plates and sequenced, the gene library of DEV was constructed by shotgun way. A DNA fragment containing complete ORF of nucleocapsid protein gene wasdiscovered in this library and considered as a nucleocapsid protein gene by analysis of its sequence with other avian herpesvirus gene in GeneBank. A set of primer was designed based on this fragment and used for amplifying to DEV DNA, and the amplified fragments were ligased to vector pGEM-T and transferred into E. coli.JM109. By analysis of PCR and sequencing and bioinformatics, the DNA fragment was identification as the nucleocapsid protein gene of DEV.4. Development and Application of A Polymerase Chain Reaction to Detect the Nucleocapsid Protein Gene of DEV: A set of primers was designed on account of a DNA fragment including nucleocapsid protein gene, and used for a polymerase chain reaction to DEV genome and clinical samples of infected ducks. The results indicated that a specific DNA fragment (about lOOObp) was amplified from DNA samples of two DEV strains, but not from nucleic acid samples of DHV, DHBV, E.coli, S.anatum, R.Anatipestifer and GPV. The specific DNA product was also amplified from 32 DNA samples extracted from livers or brains of the DEV infected ducks. As little as 1 pg of DEV DNA could be detected by this PCR. These results suggested that this PCR was a specific and sensitive method, which can be used for detection and diagnosis of clinical DEV infection.5. Constraction of Prokaryotic Expression Vector of the Nucleocapsid Protein Geneof DEV: In this experiment, a pair of oligonucleotide primers was designed by means of Oligo program and synthesized. By use of PCR, the nucleocapsid protein (NP) gene fragment of DEV was amplified from vector pGEM-NP supplied containing NP gene and was retrieved from 0.8% agarose gel and digested by BamHI and Hindlll , and then directly ligased to prokaryotic expression vector pET32a digested by the same enzymes. The recombinant plasmid was transferred into competent cells of E Coli DH5a. The results of the PCR and double digestion test showed that the nucleocapsid protein gene was constructed into prokaryotic expression vector pET32a ( designated as pET32-NP ).6. Expression of Nucleocapsid Protein Gene of DEV in E.Coli and Preliminary Study on the Antigenicity of Recombinant Protein: The recombinant plasmid pET32-NP, which was extracted from E.coli DH5a, was transferred into E.coli BL21 (DE3) byA small scale induce experiment is conduct to determine the behavior of recombinant plasmid. A recombinant fusion protein NP band expressed from pET32-NP was obviously at the position of 48 kD just as expected. On the basis of the observation to influencee of different induct time and IPTG concentration on the expression of fusion protein NP, the optimal induction condition was considered as 0.4mmol/L IPTG and induct time of three hours. The recombinant protein NP expressed from pET32-NP was amounted to about 21.5% of the total bacterium proteins by SDS-PAGE analysis and 43% products of the recombinant proteins were soluble. Analysis of Western Blotting and ELISA, the recombinant protein NP was manifested possessing favorable antigenicity.