人Hepassocin的原核可溶性表达与纯化

目的：构建人Hepassocin的原核表达载体，可溶性表达并纯化得到高纯度的重组人Hepassocino方法：将人Hepassocin基因克隆到原核表达载体pET40b（＋），转化大肠杆菌BL21（DE3），于28℃经0．1mmol／LIPTG诱导6h，表达Ds-bC-Hepassocin融合蛋白，经镍柱纯化可溶性融合蛋白，用肠激酶切除融合蛋白的DsbC-His标签，再用镍柱纯化分离酶切后的Hepassocin，通过超滤进一步纯化并浓缩，用Western blot验证纯化后的Hepassocin。结果：构建了pET40b-Hepassocin原核表达载体，经诱导表达、亲和层析和肠激酶切除融合标签，获得了相对分子质量约32000的可溶性高纯度蛋白，Western blot鉴定证实该蛋白为不含融合标签的重组人Hepassocin。结论：实现了人Hepassocin的原核可溶性表达，通过纯化获得了较高纯度的重组人Hepassocin，为制备其单克隆抗体，进一步研究其生物学功能奠定了基础。

Expression and Purification of Soluble Human Hepassocin in Escherichia coli

Objective： To express and purify soluble human Hepassocin in prokaryotic expression system. Methods： The gene of human Hepassocin was cloned into expression vector pET40b（＋）, and DsbC-Hepassocin fusion protein from transformed E.coli BL21 （DE3） after induction by 0.1 mmol/L IPTG at 28℃ for 6 hours was purified using Ni-NTA affinity chromatography column. Then the fusion protein was digested by enterokinase to remove extra exogenous amino acid residues followed by purification with Ni-NTA affinity chromatography again and ultrafihration. The ultimate purified product was characterized by Western blot. Results： The recombinant prokaryotic expression vector pET40b-Hepassocin was constructed successfully. Soluble and high-purity protein about 32 kD was obtained after twice Ni-NTA affinity chromatography and enterokinase digestion. Then it was confirmed by Western blot with specific antibody for Hepassocin. Conclusion： It laid a foundation for preparation of monoclonal antibody and research of the functions of Hepassocin in future.