蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证  

In Silico Cloning of EfP-0,a Novel Earthworm Fibrinolytic Enzyme Gene and Verification of its Coding Region by RT-PCR

作　　者：赵晓瑜[1] 高珊[1] 崔大岺[1] 耿风廷[1] 

机构地区：[1]河北大学生命科学学院,保定071002

出　　处：《生物工程学报》2006年第6期897-901,共5页Chinese Journal of Biotechnology

基　　金：河北省生物工程重点学科资助。~~

摘　　要：利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。There are four different types of N-terminal amino acid sequences （ F- Ⅰ -0, F- Ⅰ , F-Ⅱ , F-Ⅲ ） in the multi- components of earthworm fibrinolytic enzymes （EFE） . In GenBank 21 nucleic acid sequences of EFE have been reported. Among them, most of the N-terminal amino acid sequences belong to the F-Ⅲ type,few belong to the F-Ⅱ type. Only one is similar to the F- Ⅰ type, but none to F- Ⅰ -0. In this research we hoped to obtain the gene encoding component F- Ⅰ -0 of EFE by the bioinformatics tools. Based on the N-terminal amino acid sequence VVGGSDTTIGQYPHQL of the F-Ⅰ-0 type from Lumbricus rubellus, a nucleic acid sequence was obtained by in silico cloning from dbEST of Lumbricidae using the software DNAMAN. A new gene of EFE from Eisenia foetida was successfully obtained by RT-PCR using specific primers designed according to this sequence. The new gene named EfP-0 was cloned in pMAL-c2x and expressed as the fusion protein MBP-EfP- 0 in the supernatant of lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography had hydrolytic activity on casein plate. Sequencing result shows, EfP-0 has 678bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to trypsin family. It has similar amino acid composition to F- Ⅰ -0. BLAST in GenBank shows that the similarity is lower than 40% between EfP-0 gene and other EFE genes. By this we conclude that EfP-0 gene of EFE is a novel gene and it is the first time to be reported, its accession number for Genbank is DQ836917.

关 键 词：蚯蚓纤溶酶 电子克隆 Efp-0 

分 类 号：Q75[生物学—分子生物学]