烟草节杆菌02181肌酸酶的基因克隆

摘要

Objective To clone the whole sequence of creatinase gene from genomic DNA of Arthrobacter Nicotianae 02181 based on its partial sequence got previously. Methods The method of genome walking was used in this research to clone the upstream and downstream sequences adjacent to the partial sequence of creatinase gene. Then the whole sequence of creatinase gene was obtained according to the three parts and proved by BLASTx in NCBI. Results The creatinase gene obtained from Arthrobacter Nicotianae 02181,consisted of 1 254 bp (containing the stop codon TAA),which encoded 417 amino acid which had the theoretical molecular mass of 46 377 Da,was never reported before. The resulta of BALSTx in NCBI showed that the identity and positive of amino acid were 80％ (332/417) and 87％ (365/417) respectively between the creatinase gene and that from Arthrobacter sp. FB24 among all those bacteria which produced creatinase Conclusion The whole sequence of creatinase gene from Arthrobacter Nicotianae 02181 was cloned and prepared to express in the next step. Also,it may be helpful to produce the creatinine detection kit.%目的 在已知烟草节杆菌02181肌酸酶基因部分序列的基础上,从烟草节杆菌02181基因组DNA中克隆得到肌酸酶全基因.方法 根据已得到的肌酸酶部分序列,利用基因组步移技术,分别克隆得到肌酸酶基因已知部分序列的上、下游序列,再将3段序列拼接,得到肌酸酶全基因,并在NCBI上检索证实.结果 得到的烟草节杆菌02181肌酸酶基因的全长序列,在NCBI上检索后证实为一新基因,该基因全长1 254 bp(含终止密码子TAA),为一完整的开放读码框架(ORF),编码417个氨基酸,理论分子量为46 377 Da,并与多种不同来源的肌酸酶有着较高的同源性.其中,与Arthrobacter sp.FB24来源的肌酸酶氨基酸序列的一致性达到了80%(332/417),同源性则达到了87%(365/417).结论 成功地克隆了烟草节杆菌02181肌酸酶基因,为今后的基因表达奠定了基础,也为肌酐测定试剂的国产化迈出了坚实的一步.