睾丸酮丛毛单胞菌的分离和鉴定及3α-羟类固醇脱氢酶在大肠杆菌中的克隆表达

目的:从自然界中分离鉴定产3α-羟类固醇脱氢酶(3α-hydroxysteroid dehydrogenase,3α-HSD)的睾丸酮丛毛单胞菌,并在大肠杆菌(E.coli.)中克隆表达3α-HSD.方法:将池塘泥浆样品接种到以雄酮为唯一碳源的培养基中,对分离到的菌株根据形态学和生理生化特征进行初步鉴定.为进一步鉴定该菌株,根据睾丸酮丛毛单胞菌中的3α-HSD的基因设计引物,PCR扩增细菌基因组DNA,将扩增片段插入到载体pET15b中,构建重组质粒pET15b,并在E.coli. BL21(DE3)pLysS中进行诱导表达,对表达得到的蛋白进行鉴定并测定其酶活力.结果:一系列生化反应和培养特征证实,该菌与睾丸酮丛毛单胞菌的符合率为85%,最适生长pH值为7.0,最佳生长温度为30 ℃.在E.coli. BL21(DE3)pLysS中经诱导表达得到了相对分子质量约为29×103 具有酶活性的融合蛋白,可催化雄酮(3α-羟类固醇)脱氢.结论:从池塘泥浆中分离到一株产3α-HSD的睾丸酮丛毛单胞菌,并在E.coli.中表达得到了具有酶活性的融合3α-HSD.这为利用金属螯和亲和层析纯化融合3α-HSD,并进一步建立以其为工具酶的血清总胆汁酸酶循环测定方法奠定了基础.

Isolation and identification of Comamonas testosteroni: cloning and overexpression of 3α-hydroxysteroid dehydrogenase in E.coli.

OBJECTIVE: To isolate and identify 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) producing Comamonas testosteroni from soil, and to clone and overexpress 3alpha-HSD in E.coli. METHODS: Samples of pond mud were inoculated into cultural medium with androsterone as sole carbon source. The primary identification was performed according to the morphological observation, biochemical reaction and cultural characterization. To further identify the bacteria, a couple of primers were designed according to the 3alpha-HSD gene of Comamonas testosteroni. An 800 bp fragment containing 3alpha-HSD gene was obtained by PCR amplification. Then the PCR products were inserted into plasmids pET-15b to construct recombinant plasmids pET-15b. Afterwards the host bacteria containing recombinant plasmids pET-15b with proper orientation grew with isopropyl-beta-D-thioglactopyranoside (IPTG) induction. RESULTS: The isolated bacteria which could use androsterone as the sole carbon source had 85% consistency with Comamonas testosteroni. After 5 hours of IPTG induction, a recombinant protein about 29 x10(3) with enzyme activity was overexpressed in the host bacteria E.coli. BL21(DE3) pLysS. This protein could catalyze the dehydrogenization reaction of androsterone (3alpha-hydroxysteroid). CONCLUSION: A strain of Gramjnegative 3alpha-HSD producing Comamonas testosteroni was isolated from pond mud, and recombinant 3alpha-HSD with enzyme activity was overexpressed in E.coli. This work laid good foundation for the purification of recombinant 3alpha-HSD by metal chelate chromatography, and also for the construction of an enzymatic cycling method to measure serum total bile acids with recombinant 3alpha-HSD as the tool enzyme.