摘要
参照已知的L－山梨酮脱氢酶基因序列，合成了两个引物序列，以AcetobacterliqueficiensIF012258的染色体DNA为模板进行PCR反应，克隆得到了L－山梨酮脱氢酶基因，经酶切验证与预期结果相同，序列测定结果也与已知序列一致。采用PCR方法在此基因的两端加上了E．coRI和HindⅢ两个酶切位点，经E．coRI和HindⅢ酶切后去掉了两端的多余序列后，将此片段连接到pKKH上，经诱导无蛋白表达，采用RcaI酶切启动子端，对载体pKKH则采用Mcol酶切，使表达载体的SD序列与起始密码子ATG之间的距离减少了一个碱基，终止子端仍采用HindⅢ酶切，连接后进行转化，得到的阳性克隆经诱导后有明显的蛋白表达条带，酶活力也比对照明显增高。此工作为构建可直接利用L－山梨糖发酵产生维生素C前体2－酮基－L－古龙酸的基因工程菌打下了基础。
PCR method was used to arnplify the gene of L-sorbosone dehydrogenase with the template of chromosomal DNA of Acetbacter liqueficiens IFO12258. The product of PCR was cloned with a cloning vector pGEM-T and the correct L-sorbosone dehydrogenase gene was got after sequences determination. Restriction enzyme site E. coRI and HindⅢ was added to the end of L-Sorbosone dehydrogenase gene respechvely by PCR techniques. Then it was cloned to an expression vector pKKH direchy and after inducing with IPTG it can' t be expressed and no enzyme activity can be determined. Restriction enzyme RceaI was used to replase E.CoRI which resulted in a base number decreasing of the distance from SD sequence to the promoter After inducing, an obvious protein stripe had been seen and a prominentenzyme activity had been determined.
出处
《微生物学通报》
CAS
CSCD
北大核心
1998年第3期139-143,共5页
Microbiology China
关键词
PCR
表达载体
SD序列
克隆
表达
L-sorbosone dehydrogenase gene, PCR, Expression vector, SD sequence, Clone,Expression
