蚯蚓纤溶酶基因的克隆与序列分析  

Cloning and Sequence Analysis of Earthworm Fibrinolytic Enzyme Gene

作　　者：黎明[1] 赵明明[1] 王敏 张键[1] 杜连祥[1] 

机构地区：[1]天津科技大学生物工程学院天津市工业微生物重点实验室,天津300222

出　　处：《生物技术通讯》2006年第3期322-324,共3页Letters in Biotechnology

基　　金：天津科技大学科研基金项目(118099);天津市教委基金项目(20040805)

摘　　要：目的:获取蚯蚓纤溶酶(EFE)的基因,并对其进行序列分析。方法:从赤子爱胜蚓中提取总RNA,然后利用RT-PCR技术扩增EFE的基因,并将其插入pUCm-T载体,经PCR和酶切鉴定后进行序列测定及分析。结果:DNA序列分析表明,所克隆的EFE基因全长为738bp,其中编码区段为735bp,共编码245个氨基酸残基,成熟肽为238个氨基酸残基。与GenBank中已报道的粉正蚓的EFE序列F-Ⅲ-2的同源性最高,两者在核苷酸序列上有2处不同,即第137位(T→C)和第632位(G→T),密码子也因而分别由GTC、GGT变为GCC、GTT,导致第46位、211位的氨基酸残基分别由缬氨酸、甘氨酸变为丙氨酸和缬氨酸。结论:从赤子爱胜蚓中成功克隆了1条EFE基因F245。序列测定及同源性分析表明,蚯蚓纤溶酶F245与已报道的多条EFE基因序列具有高度同源性,且具备完整的编码区。该序列的克隆为用基因工程的方法生产单一成分的高酶活性EFE奠定了基础。Objective： To obtain earthworm fibrinolytic enzyme （EFE） gene and analyse its sequence. Methods： The total RNA from Eisenia foetida was extracted and the EFE gene was amplified by RT-PCR, and then inserted into pUCm-T vector. The recombinant plasmid was identified by PCR and restriction enzyme digestion and then the EFE gene was sequenced and analysed. Results： DNA sequence analysis revealed that the EFE gene was 738 bp, and that the encoding fragment was 735 bp, which encoded a protein of 245 amino acid residues and 238 amino acid residues as mature peptide chain. The sequence analysis showed that there were two nucleotide mutations （T→C, G→T）, compared to the Lumbricus rubellus F-Ⅲ-2. The further amino acid analysis demonstrated that the differention were from GTC to GCC, GGT to GTY, which subsequently led to the changes of two amino acids from V→A,G→V. Conclusion： Earthworm fibrinolytic enzyme gene F245 was successfully cloned and sequenced. Sequence analysis showed that the deduced amino acids of EFE gene F245 and other ones reported shared quite high homology. F245 had the whole coding sequence of earthworm fibrinolytic enzyme, which laid a good foundation for obtaining individual component EFE of the high activity by gene engineering method.

关 键 词：赤子爱胜蚓 蚯蚓纤溶酶 克隆 序列分析 

分 类 号：Q785[生物学—分子生物学]