CONSTRUCTION AND EXPRESSION OF PROKARYOTIC EXPRESSION PLASMID OF cDNA CODING FOR GROUP II ALLERGEN OF DERMATOPHAGOIDES FARINAE

Objective To construct and express prokaryotic expression plasmid of NA coding for group Ⅱ allergen of Dermatophagoides farinae (Der f 2). W Methods The NA of Der f 2 was digested with restriction endonuclease from recombinant plasmid pMD 18T Der f 2 and was inserted into expression vector pET 32a(+) by subclone technique, then the recombinants were transferred into Escherichia coli DL21 and identified by restriction endonuclease digestion and PCR. After that, the genetically engineered bacteria which including pET 32a(+) Der f 2 plasmids were induced by IPTG, the expression product was analyzed by SDS PAGE and densitometric scanning. W Results The positive recombinant plasmids pET32a(+) Der f 2 were identified by restriction endonuclease digestion and PCR, the objective gene fragment with the size of 455 bp were acquired, in accordance with the expected results. Sequence determination analysis showed that the gene homology with Der f 2 reported in GenBank was 100%. Plasmid pET 32a(+) Der f 2 could express a specific 34 ku protein in E. coli DL21, the protein accounted for 16% of total protein of recombinant bacteria. W Conclusion The prokaryotic expression plasmids, which contain Der f 2 gene fragment of D. farinae have been successfully constructed. Plasmid pET 32a(+) Der f 2 can express specific protein in E. coli DL21.