蚯蚓纤溶酶(EFE-3D)基因的合成及其在毕赤酵母中的表达

选择毕赤酵母偏爱密码子,分成32个长约48 bp且相互配对的寡核苷酸片段,合成蚯蚓纤溶酶基因EFE-3D。寡核苷酸片段经5′磷酸化后,通过错位拼接,PCR(聚合酶链式反应)一次性完成新基因的合成并且克隆至载体pP IC 9K中。最后利用电穿孔法将新基因克隆至毕赤酵母表达系统并进行诱导表达,用免疫印记法检测表达产物,最后利用纤维平板确定其表达产物的活性为3 500 mm2/mL,1 L发酵液相当于48 m g天然蚯蚓纤溶酶活性。

Synthesis of a Gene Encoding Fibrinolytic Enzyme from Earthworm and Its Expression by Pichia pastoris

By selecting the biased codon in P.pastoris,the new gene EFE-3D encoding earthworm fibrinolytic enzyme,consisting of 748 bp,was divided into 32 oligonucleotide fragments.The 32 synthetic fragments synthesized in vitro were assembled into one complete target fragment with only one step by a PCR approach and was cloned into pPIC9K plasmid vector.The expressing plasmid containing the synthetic gene was introduced into Pichia pastoris genome by electroporation.The secreted product was detected by Western Blotting.Fibrinolytic activity of product EFE-3D on fibrin plate was(3 500) mm~2/mL,thus the activity in 1 L fermentation broth was equivalent to 48 mg EFE activity.