水蛭素衍生物基因的克隆及其在哺乳类细胞中的表达

MOLECULAR CLONING OF HIRUDIN DERIV ATIVE cDNA GENE AND EXPRESSION IN MAMMALIAN CELL

设计特异性更高的水蛭素新突变体。化学合成编码水蛭素基因，并融会了ＲＧＤ３肽编码序列，通过连接、克隆、酶切筛选、ＤＮＡ序列分析，得到真核表达质粒ｐＡＰＲ－ＨＤ，表达质粒转染ＣＨＬ细胞，经Ｇ４１８筛选，获得表达克隆。结果：细胞培液中，重组水蛭素衍生物的活性达到１０ＡＴＵ／ｍｌ，并有抗血小板凝集作用。结论：水蛭素新突变体具有预防血栓性疾病的应用前景，但其表达水平和稳定性有待于进一步提高。

PURPOSE In order to design new hirudin variant which has more specific anticoagulation effect.METHODS The gene encoding hirudin was chemically synthesized and fused with sequence of 3 peptide RGD.The coding sequence of hirudin derivative consisted of 220 bp. Ten oligonucleotides were synthesized.N-terminal fregment was composed of six oligonucleotides,C-terminal fregment was composed of four oligonucleotides, The two fregments were linked at Kpn I site and then ligated between EcoR I and BamH I sites of pUC 19. The complete gene of hirudin derivation was recombined with a eukaryotic expression plasmid pAPRM8 containing CMV promote. Chinese hamster lung cells was transfected by the recombinant plasmid and screened by G418, the expression clone was picked up. The activity of hirudin was determined.RESULTS The activity of recombinant hirudin derivation in the culture medium reached 10 ATU/ml and a certain inhibitory effect on platelet condensation was also observed.CONCLUSIONS The new hirudin variant has potent application prospects in the prevention of thrombosed diseases, but its expression level and stability need to be futher improved.