水蛭抗凝多肽基因在大肠杆菌中的表达

Geme expression of an anticoagulant peptide of Leech in Escherichia Coli

目的 :得到具有生物活性的水蛭素。方法 :应用我国所产的吸血水蛭 ,通过逆转录酶和 PCR技术 ,获取水蛭素基因 ,将此基因克隆进质粒 p GEM- 3Zf( + )中 ,应用所得的水蛭素基因克隆进我国特有的表达载体 p BV2 2 0 中 ,并获得水蛭素的表达载体 p BV- HV菌株。结果 :该菌株通过发酵实验 ,已获得高效表达 ,并得到具有生物活性的水蛭素。结论 :菌体粗提液经乙醇分级沉淀 ,分子筛柱层析和 HPLC纯化得到纯品水蛭素 ,电泳结果证明是一条带。

Using the medicinal Leech of our country,Hirudo nipponia whitman,we have get a gene of an anticoagulant peptide Hirudin through reverse transcriptase and PCR technology,then cloned the gene into plasmid pGEM-3Zf(+).Restriction analysis of the recombinant plasmid was carried out and the restriction map was constructed.We had also measured and idetified the sequences of the plasmid and proved that the gene is hirudin gene.A expression vector with the hirudin gene had been constructed using a specific expression vector PBV 220 .The expression Vector of hirudin is PBV-HV.Shifting the culture temperature of E.coli:DH5 α from 30℃ to 42℃ 1ed to a high level expression of Hirudin.The inhibitory activity of the total soluble proteius of E.coli:DH5 α toward thrombin was evaluated by using a chromogenic thrombin substrate.We had finished the purification of hirudin through alcohol multiprecipitation,column chromatography of molecular sieve and HPLC.The result of SDS-PAGE showed a distinct band of 7000 Da.