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利用分子克隆的方法扩增与克隆睾丸酮丛毛单胞菌ATCC 11996的3α-HSD基因,构建以pET-15b为载体的表达工程菌,IPTG诱导蛋白表达,通过优化表达菌的最佳表达条件得到最大表达量的目的蛋白。诱导后将工程菌裂解,通过硫酸铵沉淀,Ni-NTA亲和层析,分子筛分离进行纯化,并测定纯化后蛋白的酶活性。结果表明:1)克隆获得了睾丸酮丛毛单胞菌3α-HSD基因并实现了异源表达;2)工程菌的最佳表达条件为:37℃培养,0.6mmol/L IPTG诱导4h,培养基中Zn2+终浓度为0.1mmol/L;3)表达工程菌裂解后用60%硫酸铵沉淀,亲和层析和分子筛分离后得到了纯度高达99%的目的蛋白,纯化后的蛋白保持酶活性。本试验成功克隆表达了睾丸酮丛毛单胞菌3α-HSD基因,建立了3α-HSD的高效表达与纯化方法,为相应抗体的制备与水资源中的类固醇激素检测方法的建立提供基础。
The 3α-HSD gene of Comamonas testosteroni ATCC 11996 was amplified and cloned by using the method of molecular cloning. The constructed expression vector pET-15b was used to express IPTG protein. The optimized expression conditions were obtained The maximum expression of the target protein. The engineered bacteria were lysed after the induction, purified by ammonium sulfate precipitation, Ni-NTA affinity chromatography and molecular sieve separation, and the enzymatic activity of the purified protein was measured. The results showed that: 1) 3α-HSD gene of Comamonas testosteroni was obtained by cloning and heterologous expression was achieved; 2) The optimal conditions for engineering bacteria were: 37 ℃ culture, induced by 0.6mmol / L IPTG for 4h, medium The final concentration of Zn2 + was 0.1 mmol / L; 3) After the engineered bacteria were lysed, they were precipitated with 60% ammonium sulfate, and the target protein with the purity of up to 99% was obtained by affinity chromatography and molecular sieve separation. In this study, 3α-HSD gene of Comamonas testosteroni was successfully cloned and expressed. The method of 3α-HSD expression and purification was established, which provided the basis for the preparation of corresponding antibodies and the establishment of steroid hormone detection method in water resources.