利用简并PCR克隆烟草节杆菌02181肌酸酶基因

Gene cloning of creatinase from Arthrobacter Nicotianae 02181 using degenerate PCR

目的用简并PCR从烟草节杆菌02181中扩增出肌酸酶部分序列,以期获得其全长编码基因。方法从NCBI查询已发表的肌酸酶蛋白质序列,将其递交到Block Maker服务器,利用工具软件CODEHOP设计简并引物,以烟草节杆菌02181基因组DNA为模板做PCR。结果将所得目的片段(414bp)在NCBI上BLASTx,结果表明,所得序列与不同菌种来源的肌酸酶高度同源,其中与节杆菌属肌酸酶基因的同源性最高,一致性达到了71%。结论从烟草节杆菌02181中成功克隆出了肌酸酶基因的部分序列。

Objective To clone partial sequence of creatinase of Arthrobacter Nicotianae 02181 using degenerate PCR so as to obtain its whole sequence. Methods We inquired the protein codes of the enzyme in NCBI and submitted them to Block Maker, then designed the degenerate primers of partial of the enzyme by CODEHOP. Degenerate PCR was used to obtain the sequence. Results The sequence （414 bp） we got was submitted to BLASTx of NCBI, which was similar to those creatinases from different bacteria. In all these bacteria, the identity between the obtained sequence and that from Arthrobacter sp. FB24 was 71%, indicating we got the right one. Conclusion We cloned partial sequence of creatinase from Arthobacer nicotianae 02181.