选用大肠杆菌Ｌ－门冬酰胺酶（ＡＳＰｓⅡ）高效表达质粒ｐＫＡ作为融合表达载体，将水蛙素Ⅲ（ＨＶ３）人工合成编码基因插入 ｐＫＡ质粒的 ＡＳＰｓⅡ编码基因 ａｎｓＢ中，使 ＡＳＰｓ Ⅱ１Ｎ端的 ８９个氨基酸残基通过甲硫氨酸残基与 ＨＶ３形成融合蛋白，从而构建成 ＡＳＰｓⅡ－ＨＶ３融合蛋白表达质粒 ｐＫＡＨ，通过基因操作将该质粒的低拷贝数复制原长更换成 ｐＵＣ高拷贝数复制原点，进而构建成ＡＳＰｓⅡ－ＨＶ３融合蛋白高效表达载体 ｐＵＫＡＨ。该质粒在大肠杆菌 ＪＭ１０５宿主细胞中表达后，融合蛋白（１５． ６ ｋＤ）占细菌总蛋白 １０ ５％，该融合蛋白经 ＣＮＢｒ切割释放出活性水蛭素分子，抗凝血活力达约 ２０ ＡＴＵ／ｍｌ培养液，为以后进一步研究打下了基础。 The plasmid pKA was highly expressed in Escherichia coli L-asparaginase (ASPs Ⅱ) as the fusion expression vector, and the coding gene of HV3 was inserted into the ASPs Ⅱ coding gene ansB of pKA plasmid. The 89 amino acids The residue forms a fusion protein with HV3 through a methionine residue to construct an ASPsII-HV3 fusion protein expression plasmid pKAH. The original copy number of the low-copy-copy of the plasmid is changed to the high copy-number origin of pUC by gene manipulation. Construction of ASPs Ⅱ-HV3 fusion protein expression vector pUKAH. After the plasmid was expressed in E. coli JM105 host cells, the fusion protein (15.6 kD) accounted for 105% of the total bacterial protein. The fusion protein was cut by CNBr to release the active hirudin molecule with an anticoagulant activity of about 20 ATU / ml culture medium, laid the foundation for further study.