人分泌磷蛋白2基因的克隆、原核表达与活性检测

提取人肝癌组织RNA,通过RT-PCR扩增人SPP2成熟蛋白编码区基因并克隆至原核表达载体pET-22b（＋）,将测序正确的质粒转化大肠杆菌BL2l（DE3）,IPTG诱导重组蛋白表达,用Ni-NTA柱进行纯化,SDS-PAGE电泳及Western blot检测并证实目的蛋白的表达,通过重组蛋白对木瓜蛋白酶的抑制作用验证其生物学活性。重组质粒测序和酶切结果显示SPP2成熟蛋白基因已成功克隆到pET-22b（＋）。IPTG诱导重组菌后有25kDa大小的目的蛋白表达。优化诱导表达条件,获得可溶性表达的目的蛋白,纯化后纯度达90%,western blot表明其具有His标签抗原活性。重组SPP2成熟蛋白可抑制木瓜蛋白酶的水解作用（酪蛋白为底物）,重量抑制比为1∶3.1,抑制比活性为2511U/mg。

Cloning,prokaryotic expression of recombinant human secreted phosphoprotein 2 and its activity determination

The target gene encoding the mature protein of human SPP2 was obtained by RT-PCR,then cloned into the prokaryotic expression vector pET-22b（＋）.The recombinant plasmid pET-22b（＋）-SPP2 was transformed into E.coli BL21（DE3） where it was induced to express protein by IPTG.The recombinant protein was purified by Ni-NTA column chromatography and detected and confirmed by SDS-PAGE as well as western blot.Its biological activity was evaluated by inhibiting the protease activity of papain.Sequence and restriction analysis reveal that the target gene encoding the mature protein of human SPP2 was cloned into pET-22b（＋）.After induction by IPTG,there was a band of protein with a molecular weight of 25kDa detected on SDS-PAGE and soluble expression was obtained by optimizing the conditions of expression.The purity of the target protein was beyond 90% and its His tag antigen activity was examined by western blot.The recombinant protein plays a role of inhibiting the protease activity of papain（casein as substrate）.Its weight ratio was about 1 ∶ 3.1 and its inhibition specific activity reached 2511U/mg.