摘要
Lumbrukinase gene from earthworm ( L.bimastus ) was obtained by RT\|PCR. The product, PI 239 , was sequenced and analyzed by biology programs and database. The gene including signal peptide coding sequence was cloned into an eukaryotic vector and the clones were obtained by transferring into BHK cells. The gene was fused with EGFP gene at C\|terminal to be detected conveniently by its fluorescence. The lumbrukinase gene PI 239 has 852 nucleotides that code for 239 amino acid residues as mature peptide chain. The N terminal of PI 239 shares certain homology with those known lumbrukinase. The enzyme contains relative more acidic amino acid residues, and has homology to serine protease. It belongs to the acidic protein, serine protease. Conformation prediction indicates that its secondary structure mainly consists of β sheet. It has two super secondary structure motifs with the active sites Asp188 and Ser189 in between. The DNA and mRNA of the whole gene could be detected in BHK clones, but no recombinant protein detected. Under cofocal microscope, the cells transferred with fused gene showed fluorescence indicating that the fused protein was expressed. In addition, the cells containing the fused gene died soon while most of the control cells were still alive. It seemed that the protein could be expressed in BHK cells as a cytotoxin, though at a low level.
Lumbrukinase gene from earthworm ( L.bimastus ) was obtained by RT\|PCR. The product, PI 239 , was sequenced and analyzed by biology programs and database. The gene including signal peptide coding sequence was cloned into an eukaryotic vector and the clones were obtained by transferring into BHK cells. The gene was fused with EGFP gene at C\|terminal to be detected conveniently by its fluorescence. The lumbrukinase gene PI 239 has 852 nucleotides that code for 239 amino acid residues as mature peptide chain. The N terminal of PI 239 shares certain homology with those known lumbrukinase. The enzyme contains relative more acidic amino acid residues, and has homology to serine protease. It belongs to the acidic protein, serine protease. Conformation prediction indicates that its secondary structure mainly consists of β sheet. It has two super secondary structure motifs with the active sites Asp188 and Ser189 in between. The DNA and mRNA of the whole gene could be detected in BHK clones, but no recombinant protein detected. Under cofocal microscope, the cells transferred with fused gene showed fluorescence indicating that the fused protein was expressed. In addition, the cells containing the fused gene died soon while most of the control cells were still alive. It seemed that the protein could be expressed in BHK cells as a cytotoxin, though at a low level.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2002年第6期776-779,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家高技术研究发展计划 (863 )生物技术领域资助 (No .10 2 0 8 0 2 0 3 )~~
关键词
蚓激酶
克隆
BHK细胞
作用
lumbrukinase, serine protease, protein PI 239 , eukaryotic expression
