地鳖虫纤溶活性蛋白cDNA序列克隆与毕赤酵母表达

依据已报道的地鳖虫成熟肽cDNA序列设计引物,通过RT-PCR法从地鳖虫(Eupolyphage sinensis Walker)中克隆得到675 bp地鳖虫纤溶活性蛋白 (fibrinolytic protein,EFP)成熟肽编码序列.将此片段克隆到表达载体pPICZα-A中,转化毕赤酵母GS115,甲醇诱导表达得到重组表达蛋白,经SDS-PAGE电泳和活性鉴定,表明重组EFP在毕赤酵母中均获得表达,重组表达蛋白相对分子质量为28.2 kD,表达产物分子质量与理论分子质量相符.重组蛋白在毕赤酵母中以分泌形式表达,具有纤溶活性.

Cloning and Expression of Eupolyphaga sinensis Walker Fibrinolytic Protein cDNA in Pichia pastoris

The primers were designed corresponding to Eupolyphaga sinensis Walker fibrinolytic protein (EFP) cDNA sequence which has been reported. The cDNA sequence encoding fibrinolytic enzyme was cloned from fresh body of Eupolyphaga sinensis Walker by RT-PCR,and then was blasted against other fibrinolytic enzyme cDNA sequences in the Genbank. The analysis of the sequence data indicated that the codeing region of cDNA fragment, which encoded 224 amino acid residues,was about 675 bp in size. The amplified cDNA fragment was cloned into the eukaryotic expression vector pPICZα-A, to produce the expression vector pPICZα-A-EFP. The re-combinant plasmid was trabsformed into P. pastoris. EFP fusion protein was obtained after the addition of methanol into the growth media. SDS-PAGE analysis revealed that the EFP was expressed . A protein band of 28. 2 kD appeared on SDS-PAGE gel. rEFP was secreted into the culture medium and was able to dissolve artificial fibrin plates.