高温酸性磷酸酶acp基因的克隆、表达和功能的初步鉴定

Cloning,Expression and Identification of a Thermostable Acid Phosphatase Gene

从高温塘泥(温度在40～85℃之间)中分离到一组嗜热微生物,提取环境基因组DNA,通过随机测序,发现许多基因与Thermus属的DNA相似性很高,根据Genbank上Thermus thermophilusHB8基因组序列中推定的酸性磷酸酶基因(TTHA1616),设计引物,PCR扩增得到一段798bp的DNA序列,测序结果与Thermus thermophilusHB8基因组中酸性磷酸酶基因相似性达98%,含有完整的酸性磷酸酶保守区,于是将该基因在大肠杆菌中表达,得到一条约33KD的特异蛋白条带,酶活检测证实该推定蛋白具有酸性磷酸酶活力。

A group of thermophilic microorganisms were isolated from the mud with a tempreture of 40-85℃. By extracting the genomic DNA and sequencing randomly,we found there was very high identity between these sequences and the genomic sequence of Thermus bacteria. According to the putative acid phosphatase gene sequence （TTHA1616）of Thermus thermophilus HB8 from Genbank,we designed primers and amplified the sequence by PCR. A 798 bp DNA sequence with putative entired acid phosphatase conserved domain,98% similar to that of TTHA1616 from Thermus thermophilus,was gotten. Then we expressed it in E.coli,and got a 33KD protein which we deduced that it had the activity of acid phosphatase.