【摘要】 参照文献上的2，5-二酮基-D-葡萄糖酸（简称2，5-DKG）还原酶II基因序列，合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点，抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应，克隆得到2，5-DKG还原酶II基因，酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2，5-DKG还原酶II基因用EcoRI和BamHI内切酶切下，连接到pBV220载体上，构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力，测序发现基因的3’末端的原PCR引物外少合了一个碱基，终止密码子发生移码突变而消失。此外在5’端的启始密码子ATG前有三个碱基与pBV220载体上的SD序列发生配对。据此，重新设计和合成了PCR引物，并用pBV220和pBL4载体构建了两个表达载体。42℃诱导表达均得到了稳定的表达条带和较高的酶活力．更多还原

【Abstract】 PCR method was used to amplify the gene of 2,5-diketo-D-gluconate reductase II with the template ofchromosomal DNA from Corynebacterium sp. Strain SCB3058. Restriction enzyme sequence FdcoR I and BamH Iwas added to the end of 2,5--DKG reductase gene respectively by PCR techniques.The gene was cloned with acoloning vector pGEM-T and expressed with an expression vector pBV220. After induced at 42℃, no exactprotein was observed. Sequence determination found that one base was lost in PCR primer which led to the lossof end code, also three bases before ATG combine to other three bases in SD sequences which affected theexpression. After correcting the two mistake by PCR method, we got the right 2,5--DKG reductase gene andexpressed it in vector PBV220 pBL4 successfully.更多还原