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. 2003;27(2):189–196. doi: 10.1023/A:1025784712265

High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris

Ping Qian 1, Xiangmin Li 1, Guangzhi Tong 2, Huanchun Chen 1,✉
PMCID: PMC7089132  PMID: 14501197

Abstract

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5′-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZαA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.

Keywords: codon bias, gene modification, high-level expression, ORF6 gene, Pichia pastoris, PRRSV

References

  • 1.Andreyev V.G., Scherbakov A.G., Pylnov V.A., Gvsev A.A. Proceedings of the International Symposium on PRRS and Aujeszky's Disease, Genetic heterogeneity of PRRSV in Russia. France: Ploufragan; 1999. pp. 211–212. [Google Scholar]
  • 2.Buckholz R.G., Gleeson M.A. Biotechnology. 1991;9:1067–1072. doi: 10.1038/nbt1191-1067. [DOI] [PubMed] [Google Scholar]
  • 3.Cavanagh D. Arch Virol. 1997;142:629–633. [PubMed] [Google Scholar]
  • 4.Clare J.J., Romanos M.A., Rayment F.B., Rowedder J.E., Smith M.A., Sreekrishna K., Henwood C.A. Gene. 1991;105:205–212. doi: 10.1016/0378-1119(91)90152-2. [DOI] [PubMed] [Google Scholar]
  • 5.Conzelmann K.K., Visser N., Van Wvensel P., Thiel H.J. Virology. 1993;193:329–339. doi: 10.1006/viro.1993.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Cregg J.M., Vedvick T.S., Raschke W.C. Biotechnology. 1993;11:905–910. doi: 10.1038/nbt0893-905. [DOI] [PubMed] [Google Scholar]
  • 7.Cregg J.M., Barringer K.J., Hessler A.Y., Madden K.R. Mol Cell Biol. 1985;5:3376–3385. doi: 10.1128/mcb.5.12.3376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Cregg J.M., Madden K.R., Barringer K.J., Thill G.P., Stillman C.A. Mol Cell Biol. 1989;9:1316–1323. doi: 10.1128/mcb.9.3.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Dea S., Gagnon C.A., Mardassi H., Pirzazdeh B., Rogan D. Arch Virol. 2000;145:659–688. doi: 10.1007/s007050050662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gagnon C.A., Dea S. Can J Vet Res. 1998;62:110–116. [PMC free article] [PubMed] [Google Scholar]
  • 11.Gellissen G., Hollenberg C.P. Gene. 1997;190:87–97. doi: 10.1016/s0378-1119(97)00020-6. [DOI] [PubMed] [Google Scholar]
  • 12.Bennetzen J.L., Hall B.D. J Biol Chem. 1982;257:3026–3031. [PubMed] [Google Scholar]
  • 13.Kreutz L.C., Mengeling W.L. Vet Microbiol. 1997;59:1–13. doi: 10.1016/s0378-1135(97)00142-9. [DOI] [PubMed] [Google Scholar]
  • 14.Loemba H.D., Mounir S., Mardassi H., Archambault D., Dea S. Arch Virol. 1996;141:751–761. doi: 10.1007/BF01718333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mardassi H., Mounir S., Dea S. Arch Virol. 1995;140:1405–1418. doi: 10.1007/BF01322667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Meng X.J., Paul P.S., Halbur P.G., Lum M.A. Arch Virol. 1995;140:745–755. doi: 10.1007/BF01309962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Mengeling W.L., Lager K.M., Vorwald A.C. J Vet Diagn Invest. 1995;7:3–16. doi: 10.1177/104063879500700102. [DOI] [PubMed] [Google Scholar]
  • 18.Meulenberg J.J., Hulst M.M., de Meijer E.J., Moonen P.L., den Besten A., de Kluyver E.P., Wensvoort G., Moormann R.J. Virology. 1993;192:62–72. doi: 10.1006/viro.1993.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Meulenberg J.J., Petersen-den Besten A., de Kluyver E.P., Moormann R.J., Schaaper W.M., Wensvoort G. Virology. 1995;206:155–163. doi: 10.1016/S0042-6822(95)80030-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Meulenberg J.J., Bos-de Ruijter J.N., van de Graaf R., Wensvoort G., Mooramann R.J. J Virol. 1998;72:380–387. doi: 10.1128/jvi.72.1.380-387.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Nelson E.A., Christopher-Hennings J., Benfield D.A. J Vet Diagn Invest. 1994;6:410–415. doi: 10.1177/104063879400600402. [DOI] [PubMed] [Google Scholar]
  • 22.Plana Duran J., Climent I., Sarraseca J., Urniza A., Cortes E., Vela C., Casal J.I. Virus Genes. 1997;14:19–29. doi: 10.1023/a:1007931322271. [DOI] [PubMed] [Google Scholar]
  • 23.Rottier P.J., Welling G.W., Welling-Wester S., Niesters H.G., Lenstra J.A., van der Zeijst B.A. Biochemistry. 1986;25:1335–1339. doi: 10.1021/bi00354a022. [DOI] [PubMed] [Google Scholar]
  • 24.Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  • 25.Scorer C.A., Buckholz R.G., Clare J.J., Romanos M.A. Gene. 1993;136:111–119. doi: 10.1016/0378-1119(93)90454-b. [DOI] [PubMed] [Google Scholar]
  • 26.Sharp P.M., Tuohy T.M., Mosurski K.R. Nucleic Acids Res. 1986;14:35125–35143. doi: 10.1093/nar/14.13.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Snijder E.J., Meulenberg J.J. J Gen Virol. 1998;79:961–979. doi: 10.1099/0022-1317-79-5-961. [DOI] [PubMed] [Google Scholar]
  • 28.Sreekrishna K., Nelles L., Potenz R.H., Cruze J., Mazzaferro P., Fish W., Holden K., Phelps D., Wood P. Biochemistry. 1989;28:4117–4125. doi: 10.1021/bi00435a074. [DOI] [PubMed] [Google Scholar]
  • 29.Sreekrishna K., Brankamp R.G., Kropp K.E., Blankenship D.T., Tsay J.T., Smith P.L., Wierschke J.D., Subramaniam A., Birkenberger L.A. Gene. 1997;190:55–62. doi: 10.1016/s0378-1119(96)00672-5. [DOI] [PubMed] [Google Scholar]
  • 30.Tong G.Z., Qiu H.J., Zhou Y.J. Adv Nat Sci. 2000;2:147–153. [Google Scholar]
  • 31.Tschopp J.F., Brust P.F., Cregg J.M., Stillman C.A., Gingeras T.R. Nucleic Acids Res. 1987;15:3859–3876. doi: 10.1093/nar/15.9.3859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yang S.X., Kwang J.K., Laegreid W. Arch Virol. 1998;143:601–612. doi: 10.1007/s007050050316. [DOI] [PubMed] [Google Scholar]
  • 33.Zeman D., Neiger R., Yaeger M., Nelson E., Benfield D., Leslie-Steen P., Thomson J., Miskimins D., Daly R., Minehart M. J Vet Diagn Invest. 1993;5:522–528. doi: 10.1177/104063879300500404. [DOI] [PubMed] [Google Scholar]

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