膜联蛋白A5 cDNA的克隆及其在大肠杆菌中的表达

cDNA cloning and expression of Annexin A5 in E.coli

目的 :克隆人膜联蛋白 A5 c DNA并在大肠杆菌系统内作表达。 方法 :利用 RT- PCR技术从人胎盘组织的总 RNA扩增膜联蛋白 A5的 c DNA序列 ,将该基因插入 GST融合表达载体 p GEX- 5 T,在 tac启动子控制下 ,IPTG诱导表达 GST融合蛋白。以亲合层析法纯化表达的融合蛋白 ,用 KPTT法验证抗凝血活性。结果 :凝胶电泳显示 PCR扩增产物的长度为 978bp,重组质粒测序结果表明 ,插入片段与人膜联蛋白 A5的序列完全一致。在 IPTG诱导下 ,K80 2重组菌高效表达出相对分子质量为 5 80 0 0的融合蛋白 ,表达量占菌体总蛋白的 2 6 %。纯化蛋白具有很强的抗凝血活性。 结论 :成功克隆人膜联蛋白

Objective:To clone and prokaryotically express human annexin A5(AnxA5) gene.Methods: The encoding sequence of human AnxA5 gene was amplified from human placenta total RNA by RT PCR and was inserted into the GST fusion expression vector pGEX 5T.The expression of fusion protein GST AnxA5 was induced by IPTG and the products were purified.The anticoagulant activity was assayed using modified kaolin partial thromboplastin time (KPTT).Results: Th e PCR product was 978 bp in size,which was consistent with the expected size of AnxA5; the sequence of insert was corresponded with the published encoding se quence of human AnxA5.Plasmid pGEX5T AnxA5 was transformed into E.coli K 802 and a new protein with a relative molecular weight of about 58 000 was induc ed after adding IPTG to the culture,which amounted to about 26% of the total ba cterial protein.Then the protein was purified by affinity chromatography.KPTT assays showed that the purified protein had high anticoagulant activity.Conclusion: The encoding sequence of human AnxA5 gene is successfully cloned into the GST fusion expression vector pGEX 5T.