Serving the Society Since 1986
Advanced Search
Volume 24 Issue 3
June 2009
    Citation: Jian-qiang LI, Ji-xing LIU, Xi LAN, Jie CHENG, Run WU, Zhong-Zi LOU, Xiang-ping YIN, Xue-rui LI, Bao-yu LI, Bin YANG, Zhi-yong LI. Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX* .VIROLOGICA SINICA, 2009, 24(3) : 179-186.  http://dx.doi.org/10.1007/s12250-009-2982-y
    shu

    Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX*

    cstr: 32224.14.s12250-009-2982-y
    • 1.

      College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

    • 2.

      Key laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

    • Corresponding author: Ji-xing LIU, liujixing@hotmail.com
    • Received Date: 31 July 2008
      Accepted Date: 08 January 2009
      Available online: 01 June 2009

      Fund Project: National High-tech Development Research Program of China 2006AA02Z440National Basic Research Program 2004CCA00500

    • The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM，M and N genes open reading frame (ORF) of DX were 4 152, 231，681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China，and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.
    • 加载中

      1. Bos E C, Luytjes W, vander Meulen H V, et al. 1996. The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology. 218: 52-60.
          doi: 10.1006/viro.1996.0165

      2. Chen J F, Feng L, Shi H Y, et al. 2007. Genetic variation and prolaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain. Chin J Prev Vet Med, 29: 856-860. (in Chinese)

      3. Cheng Q H, Niu X Y. 1992. Investigation on the porcine epidemic diarrhea prevalent on Qinhai, Chin Qinhai J Anim Vet Sci, 22: 22-23. (in Chinese)

      4. DeBouck P, Callebaut P, Pensaert M. 1982. Prevalence of the porcine epidemic diarrhea (PED) virus in the pig population of different countries, Proc Int Congr Pig Vet Soc, 7: 53.

      5. Duarte M, Laude H. 1994. Sequence of the spike protein of the Porcine epidemic diarrhea virus. J Gen Virol, 75: 1195-1200.
          doi: 10.1099/0022-1317-75-5-1195

      6. Egberink H F, Ederveen J, Callebaut P. 1988. Characte-rization of the structural proteins of porcine epidemic diarrhea virus, strain CV777. Am J Vet Res, 49: 1320-1324.

      7. Fan J H, Li Y J. 2005. Cloning and sequence analysis of the M gene of Porcine epidemic diarrhea virus LJB/03. Virus Genes. 30: 69-73.
          doi: 10.1007/s11262-004-4583-z

      8. Ge J W, Li B X, Tang L J, et al. 2006. Cloning and sequence analysis of the N gene of porcine epidemic diarrhea virus LJB/03. Virus Genes 33: 215-219.
          doi: 10.1007/s11262-005-0059-z

      9. Hofmann M, Wyler R. 1987, Serologic study of the occurrence of epizootic viral diarrhea in swine in Switzer-land, Schweiz Arch Tierheilkd, 129: 437.

      10. Kang T J, Seo J E, Kim D H, et al. 2005. Cloning and sequence analysis of the Korean strain of spike gene of Porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants. Protein Expression and Purification. 41: 378-383.
          doi: 10.1016/j.pep.2005.02.018

      11. Lee H G, Yeo S H. 2003. Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99. Virus Genes. 26: 207-212.
          doi: 10.1023/A:1023447732567

      12. Master P S. 2006. The molecular biology of coronaviruses. Adv Virus Res, 66: 193-198
          doi: 10.1016/S0065-3527(06)66005-3

      13. Mostl K, Horvath E, Burki F. 1990. Erhebungen uber porcine coronaviren in osterreich Ⅱ porcine epidemic diarrhea virus (PEDV) der schweine. Wien Tierarztl Monatsschr. 77: 10-18.

      14. Penzes Z, Gonzalez J M, Calvo E, et al. 2001. Complete Genome Sequence of Transmissible Gastroenteritis Coronavirus PUR46-MAD Clone and Evolution of the Purdue Virus Cluster. Virus Genes, 23: 105-118.
          doi: 10.1023/A:1011147832586

      15. Shibata I, Tsuda T, Mori M. 2000. Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experi-mental infection of pigs of different ages. Vet Microbiol, 72: 1732-1821

      16. Singh M. 1999. A novel internal open reading frame product expressed from a polycistronic of porcine edpidemic diarrhea virus may not contribute to virus attenuation. J Gen Virol, 80: 1959-1963.
          doi: 10.1099/0022-1317-80-8-1959

      17. Straw B E, Allaire S D, Mengeling W L, et al. 2000. In: Disease of Swine (Translated by Zhao D M, Zhang Z Q, Shen J Z). 8th ed. Beijing: The Chinese Agriculture University Press, p181-187.

      18. Tzipori S, Spradbrow P B. 1978. A cell culture vaccine against bovine ephemeral fever. Aust Vet J Jul. 54: 323-328.
          doi: 10.1111/j.1751-0813.1978.tb02480.x

      19. Yeo S G, Hernandez M, Krell P J. 2003. Cloning and sequence analysis of the spike gene of Porcine epidemic diarrhea virus Chiju99.Virus Genes. 26: 239-246.
          doi: 10.1023/A:1024443112717

      20. Wang Y, Zhang X. 1999. The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo. Virology 265: 96-109.
          doi: 10.1006/viro.1999.0025

    • 加载中

    Figures(8) / Tables(2)

    PDF

    Article Metrics

    Article views(4787) PDF downloads(17) Cited by()

    Related
    Proportional views

      Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX*

        Corresponding author: Ji-xing LIU, liujixing@hotmail.com
      • 1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
      • 2. Key laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
      • Received Date: 31 July 2008
      • Accepted Date: 08 January 2009
      Fund Project:  National High-tech Development Research Program of China 2006AA02Z440National Basic Research Program 2004CCA00500

      Abstract: The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM，M and N genes open reading frame (ORF) of DX were 4 152, 231，681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China，and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

        HTML