水蛭素基因的递归PCR合成和在大肠杆菌中的表达与分泌

Synthesis of Hirudin Gene by Recursive PCR and Its Expression and Secretion in Escherichia coli

水蛭素是凝血酶最强的特异抑制剂，可望成为预防和治疗各种血栓疾病的有效药物。根据医用水蛭（Ｈｉｒｕｄｏｍｅｄｉｃｉｎａｌｉｓ）头部水蛭素（ＨＶ－２）的氨基酸序列，通过递归ＰＣＲ，合成水蛭素基因，并重组到本实验室改建的大肠杆菌分泌型表达载体ｐＩＮ－ｏｍｐＡ－ＨＩ中，使水蛭素基因的编码序列直接位于大肠杆菌外膜蛋白Ａ信号肽序列的下游。转化大肠杆菌ＤＨ５α，筛选出重组体，经ＰＣＲ，限制酶切图谱和序列分析，结果表明所合成的水蛭素基因与设计完全一致。水蛭素基因在大肠杆菌中得到表达，重组水蛭素（ｒＨＶ－２）被分泌到细胞周质及培养基中，表达量占菌体蛋白的１９．８％，并且有明显抗凝血酶生物活性。经分离纯化，产物在质谱分析中显示基本为单一成分，分子量为６８９３．１Ｄａ，证明重组水蛭素在分泌过程得到正确加工。

According to the amino acid sequence of hirudin HV 2,six pieces of polynucleotides were designed and chemically synthesized.The whole hirudin gene was constructed with these polynucleotides by recursive PCR.Then the synthetic gene was inserted into the secretary expression vector,pIN ompA HI.The recombinant plasmids were introduced into competent cells of E.coli strain DH 5α.Its DNA sequence was determined,proved to be the same as designed.The hirudin gene was expressed in E.coli.The recombinant product was secreted into the periplasm and medium,and antithrombin activity could be detected.The expression level was 19.8% of bacteria total proteins.The product was purified by salting out,gel filtration chromatography,and ion exchange chromatography.SDS PAGE of the product showed a single band of 12k Da(as a dimer)and mass spectrometry showed the molecular weight of 6893.1 Da.It is suggested that the recombinant hirudin has been processed correctly during the secretion.