关键词: 碳青霉烯酶OXA, 巴斯德毕赤酵母, 表达, 纯化

Abstract: To amplify and identify the chromosomal DNA of Acinetobacter Baumanii which is the first isolate producing carbapenemase OXA72 in our country and to express and purify OXA72 in Pichia pastoris. MethodsThis study amplified the oxa72 gene by PCR using the extracted genomic DNA of the isolate 40 which was obtained from the clinic as a template. After the amplified gene was cloned into vector pGAPZalphaA, linearized recombinant plasmid oxa72pGAPZalphaA was transformed to Pichia pastoris GS115 by electroporation. Then the positive transformants were screened by colony PCR, western blot analysis and Nitrocefin test. Then we purified OXA72 with a nickel column. ResultsThe DNA sequence amplified by PCR from isolate 40 was identical with blaOXA72 (GenBank accession numbers AY739646). One positive transformant had a positive Nitrocefin test. SDSPAGE showed that the relative molecular weight of the recombined protein was 34?kDa43kDa. Western blotting showed the combined protein could specifically bind to the Histag antibody. The recombinant protein was obtained by purification with 95％ final purity and 40％ recovery rate. ConclusionWe successfully expressed and purified the carbapenemase OXA72 in Pichia pastoris.

Key words: Carbapenemase OXA72, Pichia pastoris, Expression, Purification