Cloning, Sequence Analysis and Proeukaryotic Expression of Peroxisomal Membrane Protein Allergen Gene from Chaetomium globosum / 中国生物工程杂志

ABSTRACT

The full length cDNA encoding peroxisomal membrane protein from Chaetomium globosum was cloned using RACE technology and the sequence in cDNA library of C. globosum in GenBank ( Accn BP099709). The 747bp full length cDNA encoding peroxisomal membrane protein allergen (pero) gene was assembled with 412bp 3'and 508bp 5'RACE products. The open reading frame was 501 bp encoding 166 amino acids. The molecular weight of the protein was 17. 5kDa and its theoretical isoelectric point was 5.75. The pero gene was amplified using specific primers of cDNA 5'and 3'untranslated region, sequence analysis indicated that the gene have 3 exons and 2 introns. ClustalX analysis revealed that amino acids sequence of pero gene from C. globosum and Neurospora crassa shared 83% high similarity. To construct pET28a-pero expressive plasmid, pero gene was inserted into pET28a expressive vector. Escherichia coli BL21 transformed by pET28a-pero plasmid was induced with IPTG. The protein expression was analyzed with SDS-PAGE. A 21kDa pero fusion protein representing the pero gene was expressed in recombinant E. coli BL21. The sequences of cDNA,DNA and deduced amino acid of the pero gene from C. globosum were submitted to GenBank (Accn AY555771, AY584753,AAS66898).