title: High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Pichia pastoris.
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 pPICZalphaA, 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.
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D003062:Codon; D012367:RNA, Viral; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D000432:Methanol