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Cloning of a Ribonucleotide Reductase Gene of the Herpes Simplex Virus Type 2 Strain G

  • Kim, Hee-Jin (Department of Biological Sciences, Konkuk University) ;
  • Lee, Si-Kyung (Department of Applied Biology and Chemistry, Konkuk University) ;
  • Byun, Si-Myung (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Lee, Hyung-Hoan (Department of Biological Sciences, Konkuk University)
  • Received : 2003.02.14
  • Accepted : 2003.04.07
  • Published : 2003.09.30

Abstract

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.

Keywords

References

  1. Bachetti, S., Evelegh, M. J. and Muirhead, B. (1986) Identification and separation of the two subunits of the Herpes simplex virus ribonucleotide reductase. J. Virol. 57, 1177-1181.
  2. Becker, Y. H. and Sarov, I. (1968) Herpes simplex virus DNA. Virol. 36, 184-192. https://doi.org/10.1016/0042-6822(68)90135-9
  3. Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. https://doi.org/10.1093/nar/7.6.1513
  4. Bollag, D. A., Rozycki, M. D. and Edelstein, S. J. (1996) Protein Methods. 2nd (eds), Wiley-Liss, New York, USA.
  5. Draper, K. G., Frink, R. J. and Wagner, E. K. (1982) Detailed characterization of an apparently unspliced ${\beta}$ herpes simplex virus type 1 gene mapping in the interior of another. J Virol. 43, 1123-1128.
  6. Galloway, D. A. and McDougall, J. K. (1981) Transformation of rodent cells by a cloned DNA fragment of Herpes simplex virus type 2. J. Virol. 38, 744-760.
  7. Galloway, D. A. and Swain, M. A. (1984) Organization of the left-hand end of the Herpes simplex virus type 2 BglII N fragment. J. Virol. 49, 724-730.
  8. Guan, C., Li, P., Riggs, P. D., and Inouye, R. (1987) Vector that facilitates expression and purification of foreign peptides in E. coli by maltose binding protein. Gene 67, 21-30.
  9. Hwang, S.H., Yoo, K. H., Moon, E. S., Cha, S. C. and Lee, H. H. (1998) Overexpression of insecticidal protein gene of Bacillus thuringiensis var. kurstaki HD1. J. Microbiol. 36, 289-295.
  10. Ju, S. -K., Park, J. -H., Na, S. -Y., You, K. -H., Kim, K. L. and Lee, M. -K. (2001) Molecular cloning and rebombinant expression of the long form of leptin receptor (Ob-Rb) cDNA as isolated from rat spleen. J. Biochem. Mol. Biol. 34, 156-165.
  11. Kang, H., Cha, S. C., Han, Y. J., Park, I. H., Lee, M. J. Byun, S. M. and Lee, H. H. (2000) High level production of glycoprotein H of HSV-1(F) using HcNPV vector system. J. Biochem. Mol. Biol. 33, 483-492.
  12. Kite, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. https://doi.org/10.1016/0022-2836(82)90515-0
  13. Langlier, Y., Dechamps, M., Buttin, G. (1978) Analysis of dCMP deaminase and CDP reductase levels in hamster cells infected with Herpes simplex viruses. J. Virol. 26, 547-553.
  14. Lee, H. H., Kang, B. J. and Park, K. J. (1998) Construction of a Baculovirus expression system using H. cunea nuclear polyhedrosis virus for eukaryotic cell. J. Microbiol. Biotechnol. 8, 676-684.
  15. McLaughlan, J. and Clement, J. B. (1983) Organization of the Herpes simplex virus type 1 t transcription unit encoding two early proteins with molecular weights of 140,000 and 40,000. J. Gen. Virol. 64, 991-1006.
  16. Malna, C. V., Riggs, P. D., Grandea, A. G., Stulko, B. E., Moran, L. S., Mcreynol, L. A. and Guan, C. (1988) A vector to express and purify proteins in E. coli by fusion and separation from maltose binding protein. Gene 74, 365-373. https://doi.org/10.1016/0378-1119(88)90170-9
  17. Sambrook, J. E., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, USA.
  18. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibition. Proc. Natl. Acad. Sci. USA 74, 5463-5467. https://doi.org/10.1073/pnas.74.12.5463
  19. Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 69, 503-517.
  20. Uh, H. S., Choi, J. H., Byun, S. M., Kim, S. Y. and Lee, H. H. (2001) Cloning, sequencing and Baculovirus-based expression of fusion-glycoprotein D gene of Herpes simplex virus type 1(F). J. Biochem. Mol. Biol. 34, 371-378.
  21. Wymer, J. P., Chung, T. D., Chang, Y. N., Hayward, G. S. and Aurelian, L. (1989) Identification of immediate-early-type-cisresponse elements in the promoter for the ribonucleotide Reductase from HSV type 2. J. Virol. 63, 2773-2784.